Small molecule drug conjugates

ABSTRACT

A binding moiety (B) for Carbonic Anhydrase IX (CAIX), the binding moiety comprising: 
     
       
         
         
             
             
         
       
     
     The binding moiety is univalent, bivalent, or multivalent. A targeted therapeutic agent may comprise the binding moiety. The invention also includes a method for treating a disease expressing elevated levels of CAIX by administering the targeted therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/004,921, filed Jun. 11, 2018, which is a divisional application ofU.S. patent application Ser. No. 15/431,310, filed Feb. 13, 2017, nowU.S. Pat. No. 10,016,511, which is a continuation application of U.S.patent application Ser. No. 15/226,439, filed Aug. 2, 2016, now U.S.Pat. No. 9,884,122, which is a continuation of PCT applicationPCT/EP2015/052214, filed Feb. 3, 2015, which claims benefit of GB1401819.6 filed Feb. 3, 2014, GB 1407530.3 filed Apr. 29, 2014, and GB1419994.7 filed Nov. 10, 2014. The contents of the above patentapplications are incorporated by reference herein in their entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (PHL-101-CON2.xml; Size:8,925 bytes; and Date of Creation: Feb. 15, 2023) is herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of small molecule targeteddrug conjugates (SMDCs) for the treatment of disease. In particular, theinvention relates to SMDCs formed of a low molecular weight ligand forbinding to Carbonic Anhydrase IX (CAIX), conjugated to a drug by acleavable linker for delivery of the drug to targeted tissues or cells.In one embodiment, the present invention relates to the application ofsuch SMDCs for the delivery of drugs that can kill or inhibit tumourcells.

BACKGROUND

The use of cytotoxic agents is at the basis of the treatment of cancerand other pathological conditions. Ideally cytotoxic agents shouldaccumulate at site of disease, sparing normal tissues. In reality thisdoes not happen. Many anticancer drugs do not preferentially accumulatein solid tumors. Indeed, it has been demonstrated in tumor-bearing micethat only a minimal portion of the injected drug reaches the neoplasticmass in comparison to the amount of cytotoxic agent that reaches healthyorgans.

The targeted delivery of highly potent cytotoxic agents into diseasedtissues is therefore desirable for the treatment of cancer and otherserious conditions. By attaching a therapeutic effector through acleavable linker to a ligand specific to a marker of disease, marker ofdisease.

-   -   the effector preferentially accumulates and acts at the intended        site of action, thus increasing the effectively applied dose        while reducing side effects. To date, monoclonal antibodies have        been considered as the ligands of choice and, indeed, research        in the field of antibody-drug conjugates (ADCs) has led to the        recent approval of two ADCs for applications in oncology:        brentuximab vedotin and trastuzumab emtansine.

However, antibodies are large macromolecules and thus often havedifficulties penetrating deeply into solid tumors. In addition, they canbe immunogenic and typically long circulation times can lead topremature drug release and undesired side effects. Moreover, theproduction of ADCs is expensive, reflecting the need for clinical-grademanufacturing of antibodies, drugs and the resulting conjugates.

The use of smaller ligands as delivery vehicles such as peptides orsmall drug-like molecules could potentially overcome some of theabovementioned problems. Their reduced size should aid tissuepenetration, they should be non-immunogenic and amenable to classicorganic synthesis thus reducing manufacturing costs. The favorableproperties of drug conjugates using folic acid or ligands againstprostate-specific membrane antigen (PSMA) as delivery vehicles have beendemonstrated and a folate conjugate has recently entered Phase IIIclinical studies. However, only a few such conjugates have beensuccessfully identified.

WO2006137092 describes the use of fluorophore-labeled Carbonic AnhydraseIX inhibitors for the treatment of cancers by inhibiting the activity ofCAIX and thereby reversing acidification of the extracellularenvironment of the tumour. There is no suggestion to use the CAIXinhibitors for targeting cytotoxic agents. Further CAIX inhibitors forthe treatment of cancer are described in WO2011098610 and WO2004048544.

The present inventors have found small molecule drug conjugates thattarget Carbonic Anhydrase IX (CAIX) expressing tumors.

SUMMARY OF THE INVENTION

According to the first aspect of the invention, therefore, there isprovided a targeted therapeutic agent comprising a compound of formula:

B-L-D  (I),

-   -   wherein.    -   B is a low molecular weight binding moiety for a Carbonic        Anhydrase;    -   D is a drug moiety; and    -   L is a linker group that undergoes cleavage in vivo for        releasing said drug moiety in an active form.

The binding moiety B suitably binds to a tumor-associated carbonicanhydrase enzyme, most preferably it binds to Carbonic Anhydrase IX(CAIX). The binding to the carbonic anhydrase is suitably selective orspecific, whereby the binding moiety B accumulates in vivo at sites,such as tumors, where carbonic anhydrase is present at elevated levels.

Alternatively or additionally, the binding moiety may bind to othercarbonic anhydrases such as Carbonic Anhydrase XII.

Suitably, the compound of Formula (I) has a molecular weight less thanabout 8,000, more suitably less than about 5000, and most suitably lessthan about 2000. In contrast to antibodies, small molecules can diffuseout of blood vessels in a matter of seconds. The distribution is notrestricted to perivascular space, but involves also deep penetrationinto tissues. This results in faster, deeper and more efficient drugtargeting by the agents of the invention.

In another aspect, the present invention provides a targeted therapeuticagent in accordance with the first aspect of the invention, for use inthe treatment of a neoplastic disease, preferably for the treatment of asolid tumor, more preferably for the treatment of renal cell carcinoma.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a targeted therapeutic agent according to thefirst aspect of the invention.

In another aspect, the present invention provides a product comprising acompound of Formula (I) as defined herein and a cleavage agent forcleaving said cleavable linker L, as a combined preparation forsequential administration in the treatment of cancer.

In another aspect, the present invention provides a method of treating aneoplastic disease, preferably a solid tumor such as renal cellcarcinoma, comprising administering an effective amount of apharmaceutical composition according to the present invention to apatient in need thereof. In embodiments, the administration of saidpharmaceutical composition is followed after a suitable interval of timeby administration of a cleavage agent for cleaving said cleavable linkerL.

Any feature described herein as suitable, optional, or preferred inrelation to any one aspect of the invention may likewise be suitable,optional or preferred in relation to any other aspect of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows chemical structures of ligand-linker-dye conjugatessynthesised for in vitro binding and in vivo targeting studies:

FIG. 2 shows structures and synthesis of small molecule drug conjugatesaccording to the present invention; “CysAspArgAsp” is identified as SEQID NO:1. “AspArgAspCys is identified as SEQ ID NO:6.

FIG. 3 shows fluorescence measurements of organ uptake ofligand-linker-dye conjugate using a ligand of the type used in theconjugates of the present invention, compared to uptake into the sameorgans of an untargeted conjugate;

FIG. 4 shows fluorescence measurements of organ uptake ofligand-linker-dye conjugate using a ligand of the type used in theconjugates of the present invention at 1 hour, 2 hours and 4 hours afteradministration of the conjugate;

FIG. 5 shows graphs of weight loss versus time for test animals treatedwith three different dosage regimens of a ligand-linker-drug conjugateaccording to the invention;

FIG. 6 shows graphs of (a) tumor volume versus time for growth of SKRC52xenografts in balb/c nu/nu mice treated 5× on 5 consecutive days withtwo different conjugates 7a and 8a according to the invention and withtwo corresponding untargeted drug conjugates, and (b) measured bodyweight change associated with the treatment;

FIG. 7 shows graphs of (a) tumor volume versus time for growth of SKRC52xenografts in balb/c nu/nu mice treated 5×on 5 consecutive days with afurther conjugate 9a according to the invention and with a correspondinguntargeted drug conjugates and with two conventional antitumor drugs,and (b) measured body weight change associated with the treatments;

FIG. 8 shows hydrolytic stability of drug conjugates 7a, 8a and 9a inPBS at pH 7.4 and 37 C as determined by liquid chromatography-massspectrometry/mass spectrometry (7a and 8a) and high-performance liquidchromatography (9a);

FIG. 9 shows structures of a monovalent ligand for CAIX and adye-conjugate thereof;

FIG. 10 shows structures of a bivalent ligand for CAIX and adye-conjugate thereof;

FIG. 11 shows the structures of a targeted bivalent drug conjugate B7according to the present invention and an untargeted control B8;

FIG. 12 shows tumor growth curves of animals injected with 8×35 nmolunconjugated ligand B2, bivalent drug conjugate B7, control conjugate B8or vehicle as control. Data represent averages±standard errors;

FIG. 13 shows a schematic representation of a member of the DNA-encodedself-assembling chemical (ESAC) library binding to its target proteinCAIX. The library displays two pharmacophores A and B and is formed byhybridization of two individually synthesised single-strandedsub-libraries A and B, resulting in a combinatorial library ofA×B=111,100 members;

FIG. 14 shows a plot of the results of high-throughput DNA sequencing(HTDS) results of reactions against CAIX for the ESAC library. The x/yplane represents the library member barcodes of sub-library A andsub-library B. and the z-axis shows the sequence counts normalised to100, cut-off level 1000 Selection conditions were high-density proteincoating (1.0 μm CAIX) and five washing steps:

FIG. 15 shows the chemical structure of untargeted IRDye 750 conjugateC6 used for flow cytometry analysis and in vivo imaging experiments;

FIG. 16 shows chemical structures and dissociation constants measured“off-DNA” by FP and SPR of synthesized monovalent and bivalentconjugates with different linker lengths;

FIG. 17 shows the chemical structure of targeted monovalent and bivalentIRDye 750 conjugates C1c and C5c used for flow cytometry analysis and invivo imaging experiments;

FIG. 18 shows flow cytometry analysis of IR-dye conjugates binding toCAIX expressing SKRC52 cells: (a) untreated cells, (b) untargetedconjugate C6, (c) targeted monovalent conjugate C1c, (d) targetedbivalent conjugate C5c;

FIG. 19 shows comparative uptake data for different organs in a mousehaving renal carcinoma of a radiolabelled anti-CAIX targeted ligandversus a radiolabelled untargeted ligand;

FIG. 20 shows a reaction scheme for the synthesis of a targetedcytotoxic drug conjugate according to the present invention having thedrug auristatin linked to a small molecule binding moiety through avaline-citrulline containing peptide linker that is cleavable byCathepsin B; and “AspArgAspCys” is identified as SEQ ID NO:6.

FIG. 21 shows data observed for mouse tumor size versus time for threedosage regimes of the drug conjugate of FIG. 20

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture,nucleic acid chemistry and biochemistry. Standard techniques are usedfor molecular biology, genetic and biochemical methods (see Sambrook etal., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., ShortProtocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.).All publications cited herein are incorporated herein by reference intheir entirety for the purpose of describing and disclosing themethodologies, reagents, and tools reported in the publications thatmight be used in connection with the invention.

Unless otherwise stated, the following definitions apply to chemicalterms used in connection of compounds of the invention and compositionscontaining such compounds.

Alkyl refers to a branched or unbranched saturated hydrocarbyl radical.Suitably, the alkyl group comprises from about 3 to about 30 carbonatoms, for example from about 5 to about 25 carbon atoms.

Alkenyl refers to a branched or unbranched hydrocarbyl radicalcontaining one or more carbon-carbon double bonds. Suitably, the alkenylgroup comprises from about 3 to about 30 carbon atoms, for example fromabout 5 to about 25 carbon atoms.

Alkynyl refers to a branched or unbranched hydrocarbyl radicalcontaining one or more carbon-carbon triple bonds. Suitably, the alkynylgroup comprises from about 3 to about 30 carbon atoms, for example fromabout 5 to about 25 carbon atoms.

Halogen refers to fluorine, chlorine, bromine or iodine, preferablyfluorine or chlorine.

Cycloalkyl refers to an alicyclic moiety, suitably having 3, 4, 5, 6, 7or 8 carbon atoms. The group may be a bridged or polycyclic ring system.More often cycloalkyl groups are monocyclic. This term includesreference to groups such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, bicyclo[2.2.2]octyl and the like.

Aryl refers to an aromatic ring system comprising 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 ring carbon atoms. Aryl may be a polycyclic ringsystem, having two or more rings, at least one of which is aromatic.This term includes reference to groups such as phenyl, naphthyl,fluorenyl, azulenyl, indenyl, anthryl and the like.

The prefix (hetero) herein signifies that one or more of the carbonatoms of the group may be substituted by nitrogen, oxygen, phosphorus,silicon or sulfur. Heteroalkyl groups include for example, alkyloxygroups and alkythio groups. Heterocycloalkyl or heteroaryl groups hereinmay have from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ringatoms, at least one of which is selected from nitrogen, oxygen,phosphorus, silicon and sulfur. In particular, a 3- to 10-membered ringor ring system and more particularly a 5- or 6-membered ring, which maybe saturated or unsaturated. For example, selected from oxiranyl,azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl,pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl,chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl,imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl,thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl,pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl,morpholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl,1,3-Dioxo-1,3-dihydro-isoindolyl, 3H-indolyl, indolyl, benzimidazolyl,cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4H-quinolizinyl,isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl,benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl,quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl,carbazolyl. [beta]-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl,chromenyl, isochromanyl, chromanyl, 3,4-dihydro-2H-isoquinolin-1-one,3,4-dihydro-2H-isoquinolinyl, and the like.

Where a substituent herein is a peptide, the peptide suitably comprisesfrom 1 to 100 amino acid residues, for example from about 2 to about 30amino acid residues.

Where a substituent herein is an oligosaccharide, the oligosaccharidesuitably comprises from 1 to 100 saccharide residues, for example fromabout 2 to about 30 saccharide residues.

“Substituted” signifies that one or more, especially up to 5, moreespecially 1, 2 or 3, of the hydrogen atoms in said moiety are replacedindependently of each other by the corresponding number of substituents.The term “optionally substituted” as used herein includes substituted orunsubstituted. It will, of course, be understood that substituents areonly at positions where they are chemically possible, the person skilledin the art being able to decide (either experimentally or theoretically)without inappropriate effort whether a particular substitution ispossible. For example, amino or hydroxy groups with free hydrogen may beunstable if bound to carbon atoms with unsaturated (e.g. olefinic)bonds. Additionally, it will of course be understood that thesubstituents described herein may themselves be substituted by anysubstituent, subject to the aforementioned restriction to appropriatesubstitutions as recognised by the skilled person.

Substituents may suitably include halogen atoms and halomethyl groupssuch as CF₃ and CCl₃; oxygen containing groups such as oxo, hydroxy,carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl andaryloyloxy; nitrogen containing groups such as amino, alkylamino,dialkylamino, cyano, azide and nitro; sulfur containing groups such asthiol, alkylthiol, sulfonyl and sulfoxide; heterocyclic groups which maythemselves be substituted; alkyl groups, which may themselves besubstituted; and aryl groups, which may themselves be substituted, suchas phenyl and substituted phenyl. Alkyl includes substituted andunsubstituted benzyl.

Where two or more moieties are described as being “each independently”selected from a list of atoms or groups, this means that the moietiesmay be the same or different. The identity of each moiety is thereforeindependent of the identities of the one or more other moieties.

Derivative. A derivative includes the chemical modification of acompound. Examples of such modifications include without limitation thereplacement of a hydrogen by a halo group, an alkyl group, an acyl groupor an amino group and the like. Derivatives further include esters andthe like that can undergo hydrolysis to release the compound.

Derivatives further includes salts of the compound. The modification mayincrease or decrease one or more hydrogen bonding interactions, chargeinteractions, hydrophobic interactions, van der Waals interactionsand/or dipole interactions.

Analog. This term encompasses any enantiomers, racemates andstereoisomers, as well as all pharmaceutically acceptable salts andhydrates of such compounds.

Target

The present invention targets Carbonic Anhydrase, in particular CarbonicAnhydrase IX (CAIX) proteins that are expressed on tumours. CAIX isover-expressed in many different forms of cancer such as glioblastoma,colorectal and breast cancer as a marker of hypoxia, while being almostundetectable in normal adult tissues, thus representing a veryattractive antitumor target. In renal cell carcinoma it is oftenconstitutively expressed and is among the best-characterizedcell-surface markers of this disease

Binding Moiety

Suitably, the binding moiety is a low molecular weight binding moiety.Thus, the binding moiety is preferably not an antibody or an antibodyfragment. Suitably, the molecular weight of the binding moiety is lessthan about 10,000, preferably less than about 3000, most preferably lessthan about 1000. In embodiments, the binding moiety (ligand) is apeptide. In other embodiments, the binding moiety (ligand) is not apeptide. The possibility to step away from antibodies and to use smallorganic or inorganic molecules as ligands allows those molecules to havecomplexity with is amenable to chemical synthesis. The core of thestructures can vary from pure organic compounds to structures that arebased on peptide scaffolds and even inorganic structures such as boronand other clusters

The binding moiety may be based on a compound that is known to bindstrongly to the target. Alternatively, the binding moiety may beidentified by one or more known screening methods for identifyingcompounds that bind selectively to the target protein of interest.

For example, improved variants of the ligands described below, or newligands for binding selectively to target proteins of interest, can befound by screening methods using modern medicinal chemistrytechnologies, e g. DNA-encoded chemical library technologies asdescribed in WO2009077173 and by R. E Kleiner et al in Chemical SocietyReviews 40 5707-5717 (2011), L. Mannocci et al. in ChemicalCommunications 47, 12747-12753 (2011) and S. Brenner et al. inProceedings of the National Academy of Sciences of the USA 89 5381-5383(1992). An example of a screening method used to identify the bestbinding moiety for CAIX from a library of 111,100 small organicmolecules is described in more detail below.

The binding moiety must tolerate attachment to the rest of the conjugatewhile maintaining binding affinity for its target. Suitably, theconjugate exhibits a binding affinity to its target (typicallyrecombinant CAIX) such that the resulting complex has K_(D) less thanabout 50 nM, more suitably less than about 30 nM, less than about 20 nM,less than 10 nM, less than nM, less than 2 nM, or less than 1 nM.

Carbonic anhydrases are thought to have a catalytic mechanism whichrelies upon an active site which contains a coordinated zinc ion.Carbonic anhydrase inhibitors such as acetazolamide and methazolamidewhich have terminal sulfonamido groups are thought to act by forming anadduct between the zinc ion and the terminal nitrogen of thesulfonamide. Accordingly, the binding moieties in the conjugatesaccording to the present invention suitably have a terminal sulfonamide(—SO₂NH₂), sulfamate (—OSO₂NH₂) or sulfamide (—NHSO₂NH₂) group. Mostsuitably, the terminal group is a sulphonamide group. Suitably, theterminal sulphonamide, sulfamate or sulfamide group is bonded to an arylgroup, for example to form an arylsulfonamido group —ArSO₂NH₂.

The aryl group in these embodiments typically has a single ring or twofused rings. The aryl group may be carbocyclic or heterocyclic and maybe substituted or unsubstituted. Typically, small substituents arepreferred such as Me, Et, OH, MeO, CF₃, F, Cl, Br, I and CN. Whether ornot the Ar group is substituted, two ring positions are taken up withthe terminal sulfonamide group and the bond to the rest of theconjugate. These two ring positions may be at any point on the Ar ring.

Suitably, the aryl group is a thiadiazolyl group. In these embodiments,the ligand suitably comprises the following terminal moiety (T1):

Suitably, the remainder of the conjugate is bonded to the thiadiazolylgroup through an amide group, whereby the binding moiety (ligand)comprises the following terminal moiety having a structure similar tothe terminal moiety of acetazolamide (T2):

In other embodiments, the above terminal moiety is modified by 4-Nmethylation of the thiadiazole group whereby the binding moiety (ligand)comprises the following terminal moiety having a structure similar tothe terminal moiety of of methazolamide (T3):

The binding moieties used in the present invention are not limited tosulfonamido derivatives. For example, coumarin ligands are also known tobind to CAIX. The skilled person using the techniques described hereinand common general knowledge will be able to identify further suitableligands for use as the binding moiety.

In embodiments, the binding moiety B may be a univalent binding moietyor a multivalent binding moiety, for example a bivalent binding moiety.The term “univalent binding moiety” refers to a binding moietycomprising a single ligand for binding to CAIX. The term “multivalentbinding moiety” refers to a binding moiety having two or more bindingligands (which may be the same or different) for binding to the targetentity. Suitably, the binding moiety is bivalent. The two or morebinding ligands are separated by suitable spacer groups on themultivalent binding moieties. The use of multivalent binding moietiescan provide enhanced binding of the binding moiety to the target.

Suitably, in these embodiments at least one of the two or more bindingligands comprises a terminal moiety

In these embodiments binding moiety is suitably a bivalent bindingmoiety comprising a first binding ligand comprising a terminal moiety asdefined above and a second binding ligand selected from the groupconsisting of ligands having a terminal moiety as defined above (anembodiment of this type having the formula B7 is shown in FIG. 11 ) andligands having the terminal group

wherein R′ is H or C1-C7 alkyl, C1-C7 alkenyl, or C1-C7 heteroalkyl,optionally substituted with one, two or three substituents, andpreferably R′ is methyl.

In embodiments of the latter type, the binding moiety suitably comprisesor consists essentially of:

wherein R is selected from the group consisting of.

wherein R′ is H or C1-C7 alkyl, C1-C7 alkenyl, or C1-C7 heteroalkyl,optionally substituted with one, two or three substituents, andpreferably R′ is methyl.

Suitably, the binding moiety has a binding affinity for CAIX such thatthe K_(D) for binding of a ligand-fluorescein isothiocyanate conjugatewherein the dye conjugate has structure as shown in FIG. 1 torecombinant CAIX in vitro as determined by fluorescence polarizationanalysis as described herein is less than about 50 nM, preferably lessthan about 20 nM, more preferably less than about 15 nM.

Linker

The linker attaches the binding moiety to the drug moiety. The linkermay be a bifunctional or a multifunctional moiety which can be used tolink one or more drug moieties and binder moieties to form the SMDC. Inembodiments, the conjugates of the present invention have a linker thatlinks one drug moiety to one binding moiety (which may be univalent ormultivalent).

The cytotoxic payloads should stably remain attached to the ligand whilein circulation, but should be released when the conjugate reaches thesite of disease.

Release mechanisms depend on a cleavable bond or other cleavablestructure that is present in in the linker. The cleavable structure maybe similar to those specific to antibodies or other small moleculeslinked to cytotoxic payloads. Indeed the nature of the ligand isindependent on that respect. Therefore we can envisage pH-dependent[Leamon, C. P. et al (2006) Bioconjugate Chem., 17 1226; Casi, G. et al(2012)J. Am. Chem. Soc., 134, 5887], reductive [Bernardes, G. J et al(2012) Angew. Chem. Int. Ed. Engl., 51. 941; Yang, J. et al (2006) Proc.Natl. Acad. Sci. USA, 103, 13872] and enzymatic release[Doronina S. O.et al (2008) Bioconjugate Chem, 19 1960; Sutherland, M. S. K. (2006) J.Biol. Chem, 281 10540]. In a specific setting, when functional groupsare present on either the ligand or payloads (e.g. thiols, alcohols)which allow the creation of a cleavable bond, a linkerless connectioncan be established thus releasing intact payloads, which simplifiessubstantially pharmacokinetic analysis. A non-exhaustive list ofmoieties, which have cleavable bonds and which may be incorporated intolinkers, is shown in the following table:

Release Linker Structure mechanism  

amide

Proteolysis ester

hydrolysis carbamate

hydrolysis hydrazone

hydrolysis thiazolidine

hydrolysis disulfide

reduction

wherein the substituents R and R^(n) in the above formulas may suitablybe independently selected from H, halogen, substituted or unsubstituted(hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl,(hetero)arylalkyl, (hetero)cycloalkyl, (hetero)cycloalkylaryl,heterocyclylalkyl, a peptide, an oligosaccharide or a steroid group.Suitably R and R^(n) are independently selected from H, or C1-C7 alkylor heteroalkyl. More suitably, R and R^(n) are independently selectedfrom H, methyl or ethyl.

Suitably, the conjugate is stable to hydrolysis. That is to say, lessthan about 10% of the conjugate undergoes hydrolysis in PBS pH7.4 at 37°C. after 24 hours, as determined by HPLC.

Accordingly, the linker suitably comprises as its cleavable bond adisulfide linkage since these linkages are stable to hydrolysis, whilegiving suitable drug release kinetics at the target in vivo, and canprovide traceless cleavage of drug moieties including a thiol group,such as DM1.

Suitably, the linker may be polar or charged in order to improve watersolubility of the conjugate. For example, the linker may comprise fromabout 1 to about 20, suitably from about 2 to about 10, residues of oneor more known water-soluble oligomers such as peptides,oligosaccharides, glycosaminoglycans, polyacrylic acid or salts thereof,polyethylene glycol, polyhydroxyethyl (meth) acrylates, polysulfonates,etc. Suitably, the linker may comprise a polar or charged peptide moietycomprising e.g. from 2 to 10 amino acid residues. Amino acids may referto any natural or non-natural amino acid. The peptide linker suitablyincludes a free thiol group, preferably a C-terminal cysteine, forforming the said cleavable disulfide linkage with a thiol group on thedrug moiety. A suitable peptide linker of this type is -Cys-Asp-Arg-Asp-(SEQ ID NO: 1).

Suitably, the linker is linked to the ligand through a 1,2,3-triazolering formed by 1,3-cycloaddition of alkyne and azide. The drug andbinding moieties are suitably linked to the 3 and 5 positions of thetriazole ring. The triazole ring may optionally be substituted at the 4position. The triazole is thought to improve binding of the ligand toCAIX. For example, the binding moieties identified above may be linkedthrough a triazole group to form the following terminal moiety of theconjugate:

More generally, the conjugates according to the present invention mayhave the following formula:

wherein: Hy is a hydrophilic moiety for improving the solubility of theconjugate, for example a hydrophilic oligomer as defined above such as apeptide group as defined above. S—S represents the cleavable disulfidebond between the drug moiety D and the linker.

Suitably, the disulfide bond is formed between a —SH group on thelinker, for example the —SH group of a cysteine residue (preferablyterminal cysteine) of the peptide and a —SH group present in the activeform of the drug D, for example the terminal —SH group of DM1. In thisway, reductive cleavage of the disulfide bond in vivo results intraceless release of the drug in its active form.

Sp are spacer groups, which may be independently selected fromoptionally substituted straight or branched or cyclic C1-C6 alkylene oralkenylene, optionally including one or more carbonyl carbons or etheror thioether O or S atoms or amine N atoms in the chain. The first Spgroup is suitably linked to the peptide residue by a terminal carbonylforming an amide linkage with the terminal amino group of the peptide,as shown for example in the formula 9a in FIG. 2 .

The triazole is optionally substituted at the 4 position by group R,whereby group R is selected from H or any of the substituent groupsdefined herein, or R is substituted or unsubstituted (hetero)alkyl,(hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, (hetero)arylalkyl,(hetero)cycloalkyl, (hetero)cycloalkylaryl, heterocyclylalkyl, apeptide, an oligosaccharide or a steroid group. Suitably R is selectedfrom H, halogen, halomethyl, or C1-C7 alkyl or heteroalkyl. Moresuitably, R is selected from H, methyl or ethyl, and most suitably R isH.

Alternatively or additionally to one or more of the linker elementsdescribed above, the linker in the conjugates of the present inventionmay comprise a cleavable peptide unit. The peptide unit sequence isspecifically tailored so that it will be selectively enzymaticallycleaved from the drug moiety by one or more proteases present on thecell surface or the extracellular regions of the target tissue. Theamino acid residue chain length of the peptide unit suitably ranges fromthat of a single amino acid to about eight amino acid residues. Numerousspecific cleavable peptide sequences suitable for use in the presentinvention can be designed and optimized in their selectivity forenzymatic cleavage by a particular tumor-associated enzyme e.g. aprotease. Cleavable peptides for use in the present invention includethose which are optimized toward the proteases MMP-1, 2 or 3, orcathepsin B, C or D. Especially suitable are peptides containing thesequence Val-Cit, which are cleavable by Cathepsin B. Cathepsin B is aubiquitous cysteine protease. It is an intracellular enzyme, except inpathological conditions, such as metastatic tumors or rheumatoidarthritis. Therefore, non-internalizing conjugates of the presentinvention produced with cathepsin B-cleavable linkers are stable incirculation until activated in pathological tissue.

In these embodiment, the linker moiety suitably further comprises,adjacent to the peptide sequence, a “self-immolative” linker portion.The self-immolative linkers are also known as electronic cascadelinkers. These linkers undergo elimination and fragmentation uponenzymatic cleavage of the peptide to release the drug in active,preferably free form. The conjugate is stable extracellularly in theabsence of an enzyme capable of cleaving the linker. However, uponexposure to a suitable enzyme, the linker is cleaved initiating aspontaneous self-immolative reaction resulting in the cleavage of thebond covalently linking the self-immolative moiety to the drug, tothereby effect release of the drug in its underivatized orpharmacologically active form. In these embodiments, the self-immolativelinker is coupled to the ligand moiety through an enzymaticallycleavable peptide sequence that provides a substrate for an enzyme tocleave the amide bond to initiate the self-immolative reaction Suitably,the drug moiety is connected to the self-immolative moiety of the linkervia a chemically reactive functional group pending from the drug such asa primary or secondary amine, hydroxyl, sulfhydryl or carboxyl group.

Examples of self-immolative linkers are PABC or PAB(para-aminobenzyloxycarbonyl), attaching the drug moiety to the ligandin the conjugate (Carl et al (1981) J. Med. Chem. 24: 479-480;Chakravarty et al (1983) J. Med. Chem. 26: 638-644). The amide bondlinking the carboxy terminus of a peptide unit and the para-aminobenzylof PAB may be a substrate and cleavable by certain proteases. Thearomatic amine becomes electron-donating and initiates an electroniccascade that leads to the expulsion of the leaving group, which releasesthe free drug after elimination of carbon dioxide (de Groot, et al(2001) Journal of Organic Chemistry 66 (26): 8815-8830). Furtherself-immolating linkers are described in WO2005/082023.

In these embodiments, the linker suitably further comprises a spacerunit linked to the binding moiety, for example via an amide, amine orthioether bond. The spacer unit is of a length that enables e.g. thecleavable peptide sequence to be contacted by the cleaving enzyme (e. g.cathepsin B) and suitably also the hydrolysis of the amide bond couplingthe cleavable peptide to the self-immolative moiety X. Spacer units mayfor example comprise a divalent radical such as alkylene, arylene, aheteroarylene, repeating units of alkyloxy (e.g. polyethylenoxy, PEG,polymethyleneoxy) and alkylamino (e.g. polyethyleneamino), or diacidester and amides including succinate, succinamide, diglycolate,malonate, and caproamide.

In yet other embodiments, the linker in the conjugates of the presentinvention may comprise a glucuronyl group that is cleavable byglucoronidase present on the cell surface or the extracellular region ofthe target tissue. It has been shown that lysosomal beta-glucuronidaseis liberated extracellularly in high local concentrations in necroticareas in human cancers, and that this provides a route to targetedchemotherapy (Bosslet, K. et al. Cancer Res. 58, 1195-1201 (1998)).

The number of drug and linker moieties per binding moiety, i.e. drugloading value, is suitably 1 to about 8, more suitably 1 or 2, and mostsuitably 1.

Drug

In one embodiment, the drug is a cytotoxic agent (other than aradioactive isotope) that inhibits or prevents the function of cellsand/or causes destruction of cells. Examples of cytotoxic agents includechemotherapeutic agents, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including synthetic analogues and derivatives thereof. Thecytotoxic agent may be selected from the group consisting of anauristatin, a DNA minor groove binding agent, a DNA minor groovealkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, apuromycin, a dolastatin, a maytansinoid and a vinca alkaloid or acombination of two or more thereof. In one embodiment the drug is achemotherapeutic agent selected from the group consisting of atopoisomerase inhibitor, an alkylating agent (eg, nitrogen mustards,ethylenimes; alkylsulfonates; triazenes; piperazines; and nitrosureas),an antimetabolite (eg mercaptopurine, thioguanine, 5-fluorouracil), anantibiotics (eg, anthracyclines, dactinomycin, bleomycin, adriamycin,mithramycin, dactinomycin) a mitotic disrupter (eg, plant alkaloids—suchas vincristine and/or microtubule antagonists—such as paclitaxel), a DNAintercalating agent (eg carboplatin and/or cisplatin), a DNA synthesisinhibitor, a DNA-RNA transcription regulator, an enzyme inhibitor, ageneregulator, a hormone response modifier, a hypoxia-selective cytotoxin(eg. tirapazamine), an epidermal growth factor inhibitor, ananti-vascular agent (eg, xanthenone 5,6-dimethylxanthenone-4-aceticacid), a radiation-activated prodrug (eg. nitroarylmethyl quaternary(NMQ) salts) or a bioreductive drug or a combination of two or morethereof.

The chemotherapeutic agent may selected from the group consisting ofErlotinib (TARCEVA

, Bortezomib (VELCADE

), Fulvestrant (FASLODEX

), Sutent (SU11248), Letrozole (FEMARA

), Imatinib mesylate (GLEEVEC

), PTK787/ZK 222584, Oxaliplatin (Eloxatin

), 5-FU (5-fluorouracil), Leucovorm, Rapamycin (Sirolimus, RAPAMUNE

), Lapatinib (GSK572016), Lonafamib (SCH 66336), Sorafenib (BAY43-9006),and Gefitinib (IRESSA

), AG1478, AG1571 (SU 5271; Sugen) or a combination of two or morethereof.

The chemotherapeutic agent may be an alkylating agent—such as thiotepa,CYTOXAN

and/or cyclosphosphamide; an alkyl sulfonate—such as busulfan,improsulfan and/or piposulfan; an aziridine—such as benzodopa,carboquone, meturedopa and/or uredopa, ethylenimines and/ormethylamelamines—such as altretamine, triethylenemelamine,triethylenepbosphoramide, triethylenethiophosphoramide and/ortnmethylomelamine; acetogenin—such as bullatacin and/or bullatacinone;camptothecin; bryostatin; callystatin; cryptophycins; dolastatin;duocarmycin; eleutherobin; pancratistatin; sarcodictyin; spongistatin;nitrogen mustards—such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide and/or uracil mustard;nitrosureas—such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and/or ranimnustine; dynemicin; bisphosphonates—such asclodronate; an esperamicin; a neocarzinostatin chromophore;aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN

, doxorubicin—such as morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and/ordeoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins—such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrnn, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites—such as methotrexate and5-fluorouracil (5-FU); folic acid analogues—such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogues—such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues—such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens—such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals—such as ammoglutethimide,mitotane, trilostane; folic acid replenisher—such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; macrocyclicdepsipeptides such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide;procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes—suchas verracurin A, roridin A and/or anguidine; urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacosine; arabinoside; cyclophosphamide; thiotepa; taxoids—such as TAXOL

, paclitaxel, abraxane, and/or TAXOTERE

, doxetaxel; chloranbucil; GEMZAR

, gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogues—such as cisplatin and carboplatin; vinblastine; platinum;etoposide; ifosfamide; mitoxantrone; vincristine; NAVELBINE

, vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoids—such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids, derivativesor combinations of two or more of any of the above.

The drug may be a tubulin disruptor including but are not limited to:taxanes—such as paclitaxel and docetaxel, vinca alkaloids,discodermolide, epothilones A and B, desoxyepothilone, cryptophycins,curacin A, combretastatin A-4-phosphate, BMS 247550, BMS 184476, BMS188791; LEP, RPR 109881A, EPO 906, TXD 258, ZD 6126, vinflunine, LU103793, dolastatin 10, E7010, T138067 and T900607, colchicine,phenstatin, chalcones, indanocine, T138067, oncocidin, vincristine,vinblastine, vinorelbine, vinflunine, halichondnn B, isohomohalichondrinB, ER-86526, pironetin, spongistatin 1, spiket P, cryptophycin 1,LU103793 (cematodin or cemadotin), rhizoxin, sarcodictyin, eleutherobin,laulilamide, VP-16 and D-24851 and pharmaceutically acceptable salts,acids, derivatives or combinations of two or more of any of the above.

The drug may be a DNA intercalator including but are not limited to:acridines, actinomycmns, anthracyclines, benzothiopyranoindazoles,pixantrone, crisnatol, brostallicin, CI-958, doxorubicin (adriamycin),actinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin,mitoxantrone, cyclophosphamide, melphalan, mitomycin C, bizelesin,etoposide, mitoxantrone, SN-38, carboplatin, cis-platin, actinomycin D,amsacrine, DACA, pyrazoloacndine, irinotecan and topotecan andpharmaceutically acceptable salts, acids, derivatives or combinations oftwo or more of any of the above.

The drug may be an anti-hormonal agent that acts to regulate or inhibithormone action on tumours—such as anti-estrogens and selective estrogenreceptor modulators, including, but not limited to, tamoxifen,raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and/or fareston toremifene and pharmaceuticallyacceptable salts, acids, derivatives or combinations of two or more ofany of the above. The drug may be an aromatase inhibitor that inhibitsthe enzyme aromatase, which regulates estrogen production in the adrenalglands—such as, for example, 4(5)-imidazoles, aminoglutethimide,megestrol acetate, AROMASIN

, exemestane, formestanie, fadrozole, RIVISOR

, vorozole FEMARA

, letrozole, and ARIMIDEX

and/or anastrozole and pharmaceutically acceptable salts, acids,derivatives or combinations of two or more of any of the above.

The drug may be an anti-androgens—such as flutamide, nilutamide,bicalutamide, leuprolide, goserelin and/or troxacitabine andpharmaceutically acceptable salts, acids, derivatives or combinations oftwo or more of any of the above.

The drug may be a protein kinase inhibitor, a lipid kinase inhibitor oran anti-angiogenic agent.

In a preferred embodiment, the drug is a maytansinoid, in particularDM1, or a tubulin disruptor. Preferably, the drug in its active formcomprises a thiol group, whereby a cleavable disulfide bond may beformed through the sulfur of the thiol group to bond the drug to thelinker moiety in the conjugates of the invention.

The drug may be used in unmodified or modified form. Combinations ofdrugs in which some are unmodified and some are modified may be used.For example, the drug may be chemically modified. One form of chemicalmodification is the derivatisation of a carbonyl group—such as analdehyde.

According to one embodiment, the drug is modified to allow theincorporation of the linker. For example, a drug comprising a hydroxylgroup may be converted to the corresponding 2-ethanethiol carbonate or2-ethanethiol carbamate thereby introducing thiol groups for disulphidelinkage as discussed above.

The drug can also be a cytokine (e.g., an interleukin, a member of theTNF superfamily, or an interferon.

SMDCs

The drug moiety of the SMDC may not be cleaved from the linker until theSMDC binds to its target cell or tissue.

In one embodiment, the SMDCs described herein are not substantiallyinternalized into a cell. Such a “non-internalizing” drug conjugate hasthe property of reacting in physiological conditions (at 37° C. and pH7) in vivo or in vitro, with binding partners on the cell surface (e.g.cell surface antigens) or in the extracellular matrix without beinginternalized in the cells by a process of active endocytosis (such asreceptor/antigen mediated endocytosis). It is possible that some of thenon-internalizing specific binding moiety could be taken upintracellularly by fluid phase endocytosis. However, the amount of fluidphase endocytosis will depend linearly on the extracellular bindingmoiety concentration and temperature and can therefore be distinguishedfrom mediated endocytosis in order to distinguish non-internalizingbinding moieties and conjugates according to the present invention.

The use of non-internalizing compounds provides advantages. For example,internalization efficiency is difficult to measure in vivo, thusremaining a “black box” for drug development. Moreover, it is difficultto ensure that all diseased cells are targeted by internalizingcompounds, especially those cells which are further away from bloodvessels. In contrast, the cleavage of the SMDCs of the present inventionin the extracellular space allows the drug to diffuse to neighboringcells and kill them. It is also envisaged that dying cells will liberatecleavage agents (e.g. cysteine or glutathione) that will activate moreof the drug from the SMDC resulting in self-amplification of the toxiceffects.

Accordingly, the linker that is used in the SMDC should be stable enoughcompared to the rate of blood clearance of the compound but labileenough compared to the residence time of the compound at the targetsite. From these considerations, a half-life of the conjugate in theregion of about 1 hour to about 50 hours—such as about 10 to about 50hours or about to about 50 hours may be acceptable, especially whenvascular tissues or cells are targeted. The half-life herein refers tothe half-life of the conjugate in mouse serum in vitro at 37° C. asdetermined by HPLC. Advantageously therefore, the SMDCs described hereinmay have improved lability and/or stability in vitro and/or in vivowhich makes them particularly suitable for controlled drug release,especially at vascular tissues, cells and tumours.

Suitably, the SMDC shows a high affinity for CAIX expressing tumors whenadministered systemically. Suitably, a tumor-to-blood concentrationratio of at least about 5:1, for example at least about 10:1 is achieved1 hour after injection of 3 nm of the conjugate into nude mice havingsubcutaneous SKRC52 tumors.

Suitably, the SMDC inhibits, retards or prevents growth of a tumour whenadministered in a therapeutically effective amount.

Treatment

The SMDCs described herein may be used to treat disease. The treatmentmay be therapeutic and/or prophylactic treatment, with the aim being toprevent, reduce or stop an undesired physiological change or disorder.The treatment may prolong survival as compared to expected survival ifnot receiving treatment.

The disease that is treated by the SMDC may be any disease that mightbenefit from treatment. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose to thedisorder. One particular disease that is applicable to treatment by thepresent invention is neoplastic disease such as cancer that can betreated via the targeted delivery of cytotoxic agents. Non-limitingexamples of cancers that may be treated include benign and malignanttumours; leukemia and lymphoid malignancies, including breast, ovarian,stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,pancreatic, prostate or bladder cancer. The disease may be a neuronal,glial, astrocytal, hypothalamic or other glandular, macrophagal,epithelial, stromal and blastocoelic disease; or inflammatory,angiogenic or an immunologic disease. An exemplary disease is a solid,malignant tumour.

The term “cancer” and “cancerous” is used m its broadest sense asmeaning the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. A tumour comprises one or morecancerous cells. Examples of cancer include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. Further examples of such cancers include squamous cellcancer (e.g., epithelial squamous cell cancer), lung cancer includingsmall-cell lung cancer, non-small cell lung cancer (“NSCLC”),adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, gastrointestinal stromal tumour(GIST), pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer. Based onestablished evidence of expression of CAIX, it is expected that thepresent invention will be suitable in particular for the treatment ofglioblastoma, lung cancer, head and neck cancer, cervical cancer,colorectal cancer, breast cancer, and, especially, renal cell carcinoma.

For the prevention or treatment of disease, the dosage of a SMDC willdepend on an array of different factors—such as the type of disease tobe treated, the severity and course of the disease, whether the moleculeis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the protein, andthe discretion of the attending physician.

The molecule may be administered to the patient at one time or over aseries of treatments. Depending on the type and severity of the disease,between about 1 ug/kg to 15 mg/kg of drug may be used as an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 ug/kg to 100mg/kg or more. An exemplary dosage of drug may be in the range of about0.1 to about 10 mg/kg of patient weight.

When treating cancer, the therapeutically effect that is observed may bea reduction in the number of cancer cells; a reduction in tumour size;inhibition or retardation of cancer cell infiltration into peripheralorgans; inhibition of tumour growth; and/or relief of one or more of thesymptoms associated with the cancer.

In animal models, efficacy may be assessed by physical measurements ofthe tumour during the treatment, and/or by determining partial andcomplete remission of the cancer. For cancer therapy, efficacy can, forexample, be measured by assessing the time to disease progression (TTP)and/or determining the response rate (RR).

Pharmaceutical Compositions

The SMDCs described herein may be in the form of pharmaceuticalcompositions which may be for human or animal usage in human andveterinary medicine and will typically comprise any one or more of apharmaceutically acceptable diluent, carrier, or excipient. Acceptablecarriers or diluents for therapeutic use are well known in thepharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition may be formulated to be administered using a mini-pump or bya mucosal route, for example, as a nasal spray or aerosol for inhalationor ingestable solution, or parenterally in which the composition isformulated by an injectable form, for delivery, by, for example, anintravenous, intramuscular or subcutaneous route. Alternatively, theformulation may be designed to be administered by a number of routes.

If the agent is to be administered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions may be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or thepharmaceutical compositions can be injected parenterally, for example,intravenously, intramuscularly or subcutaneously. For parenteraladministration, the compositions may be best used in the form of asterile aqueous solution which may contain other substances, forexample, enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

The SMDC may be administered in the form of a pharmaceuticallyacceptable or active salt. Pharmaceutically-acceptable salts are wellknown to those skilled in the art, and for example, include thosementioned by Berge et al, in J. Pharm. Sci., 66, 1-19 (1977). Saltsinclude, but are not limited, to sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

The routes for administration (delivery) may include, but are notlimited to, one or more of oral (e.g. as a tablet, capsule, or as aningestable solution), topical, mucosal (e.g. as a nasal spray or aerosolfor inhalation), nasal, parenteral (e.g. by an injectable form),gastrointestinal, intraspinal, intraperitoneal, intramuscular,intravenous, intrauterine, intraocular, intradermal, intracranial,intratracheal, intravaginal, intracerebroventricular, intracerebral,subcutaneous, ophthalmic (including intravitreal or intracameral),transdermal, rectal, buccal, vaginal, epidural, sublingual.

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for administration.Extemporaneous injection solutions and suspensions are prepared fromsterile powders, granules and tablets of the kind previously described.Exemplary unit dosage formulations contain a daily dose or unit dailysub-dose, or an appropriate fraction thereof, of the active ingredient.

Combination Therapy

A SMDC may be combined in a pharmaceutical combination formulation, ordosing regimen as combination therapy, with a second compound havingtherapeutic properties. The second compound of the pharmaceuticalcombination formulation or dosing regimen preferably has complementaryactivities to the SMDC of the combination such that they do notadversely affect each other.

The second compound may be selected from the group consisting of aprotein, antibody, antigen-binding fragment thereof, a drug, a toxin, anenzyme, a nuclease, a hormone, an immunomodulator, an antisenseoligonucleotide, an siRNA, a boron compound, a photoactive agent, a dyeand a radioisotope or a combination of two or more thereof.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein there is a time period while both (or all) activeagents simultaneously exert their biological activities.

As noted above, the SMDCs of the invention achieve optimal tumor-organratios some time after administration, when the SMDC has had theopportunity to localize at the site of the disease, while clearing fromblood and healthy organs. Thus, it would be desirable to providecontrolled release of the toxic payload from the SMDC at a controlledtime interval after administration. This can be achieved byadministering an effective amount of a cleavage agent for cleaving thelinker L at a later time point following SMDC administration, in orderto trigger an efficient release of the drug payload when suitabletumor:blood and tumor:organ ratios have been achieved. The time intervalbetween administration of the SMDC and administration of the cleavageagent may, for example, be from about 10 minutes to about 12 hours,suitably from about 30 minutes to about 6 hours, more suitably fromabout 1 hour to about 2 hours.

Thus, the combination products according to the invention include aproduct comprising a compound of Formula (I) as defined above and acleavage agent for cleaving the cleavable linker L, as a combinedpreparation for sequential administration in the treatment of cancer.

Suitably, either: (a) linker L comprises a disulphide bond and thecleavage agent comprises a reducing agent such as cysteine,N-acetylcysteine, ordithiothreitol; or (b) linker L comprises an amidelinkage and the cleavage agent comprises a hydrolase such as a protease;or (c) linker L comprises an ester linkage and the cleavage agentcomprises a hydrolase such as an esterase.

The cleavage agent is administered in an amount effective to achieve thedesired release of the toxic payload from the SMDC in vivo. For example,between about 1 ug/kg to 15 mg/kg of drug may be used as an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion.

An exemplary dosage of cleavage agent may be in the range of about 0.1to about 10 mg/kg of patient weight.

The above products for combined administration and methods of treatmentby sequential administration of drug conjugate and cleavage agent arealso applicable to antibody-drug conjugates as to conjugates in whichthe ligand is a low molecular weight entity. Thus, combination productsand methods in which the SMDC is an antibody-drug conjugate (ADC)comprising as the binding moiety an antibody or antibody fragment thatbinds selectively to CAIX are encompassed within these aspects of theinvention.

Substituents

The chemical compounds described herein may comprises substituents. Inparticular, the compounds may contain one or more hydroxy, alkylespecially lower (C₁-C₆) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, andn-hexyl and other hexyl isomers, alkoxy especially lower (C₁-C₆) alkoxy,e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen(e.g. fluoro) substituents.

Chemical Synthesis

The compounds described herein may be prepared by chemical synthesistechniques.

It will be apparent to those skilled in the art that sensitivefunctional groups may need to be protected and deprotected duringsynthesis of a compound. This may be achieved by conventionaltechniques, for example as described in “Protective Groups in OrganicSynthesis” by T W Greene and P G M Wuts, John Wiley and Sons Inc.(1991), and by P. J. Kocienski, in “Protecting Groups”, Georg ThiemeVerlag (1994).

It is possible during some of the reactions that any stereocentrespresent could, under certain conditions, be epimerised, for example if abase is used in a reaction with a substrate having an optical centrecomprising a base-sensitive group. It should be possible to circumventpotential problems such as this by choice of reaction sequence,conditions, reagents, protection/deprotection regimes, etc. as iswell-known in the art.

The compounds and salts of the invention may be separated and purifiedby conventional methods.

General Techniques

The practice of the present invention employs, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology and pharmacology, known tothose of skill of the art. Such techniques are explained fully in theliterature. See, e. g., Gennaro, A. R., ed. (1990) Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co.; Hardman, J. G.,Limbird, L. E., and Gilman, A. G., eds. (2001) The Pharmacological Basisof Therapeutics, 10th ed., McGraw-Hill Co.; Colowick, S. et al., eds.,Methods In Enzymology, Academic Press, Inc.; Weir, D. M., and Blackwell,C. C., eds. (1986) Handbook of Experimental Immunology, Vols. 1-IV,Blackwell Scientific Publications; Maniatis, T. et al., eds. (1989)Molecular Cloning. A Laboratory Manual, 2nd edition, Vols. I-III, ColdSpring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) ShortProtocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream etal., eds. (1998) Molecular Biology Techniques: An Intensive LaboratoryCourse, Academic Press; Newton, C. R., and Graham, A., eds. (1997) PCR(Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

Examples

(A) Monovalent Binding Moieties

Reference compounds having formulas 1a-6c shown in FIG. 1 were preparedfor studies of binding by ligand-linker-dye conjugates to CAIX in vitroand in vivo. Conjugates according to the present invention havingformulas 7a, 8a and 9a as shown in FIG. 2 were prepared according to thescheme in FIG. 2 and studied in vitro and in vivo as described below.Reference conjugates 7b, 8b and 9b as shown in FIG. 2 having the drugand linker moieties but no binding moiety were also prepared accordingto the scheme shown in FIG. 2 for comparative studies.

General Chemical Procedures

Proton (¹H) nuclear magnetic resonance (NMR) spectra were recorded on aBruker AV400 (400 MHz) or a Bruker AVIII500 (500 MHz) spectrometer.Carbon (¹³C) NMR spectra were recorded on a Bruker AV400 (100 MHz)spectrometer or on a Bruker AV111500 (125 MHz) spectrometer. Chemicalshifts are given in ppm using residual solvent as the internal standard.Coupling constants (J) are reported in Hz with the followingabbreviations used to indicate splitting: s=singlet, d=doublet,t=triplet, q=quartet, m=multiplet. High-resolution mass spectrometry(HRMS) spectra were recorded on a Bruker Daltronics maXis ESI-QTOF massspectrometer. Calculated and exact m/z values are reported in Daltons.

Analytical and preparative reversed-phase high-pressure liquidchromatography (RP-HPLC) were performed on an Waters Alliance HT RP-HPLCwith PDA UV detector, using a Synergi 4 μm, Polar-RP 150×10 mm column ata flow rate of 4 mL min⁻¹ with linear gradients of solvents A and B(A=Millipore water with 0.1% trifluoroacetic acid [TFA], B=MeCN).

Anhydrous solvents for reactions were purchased from Acros or Fluka.Peptide grade dimethyl formamide (DMF) for solid phase synthesis wasbought from ABCR. All other solvents were used as supplied by FisherChemicals, Merck or Aldrich in HPLC or analytical grade. IRDye750N-hydroxysuccinimidyl (NHS) ester was purchased from Licor. Alexa546 NHSester from Invitrogen, N-Boc protected(S)-1-chloromethyl-6-hydroxy-1,2-dihydrobeno[e]indole (seco CBI) fromAnthem Bioscience. DM1 was purchased from Concortis Biosystems. Allother reagents were purchased from Aldrich, Acros. ABCR or TCI and usedas supplied. All reactions using anhydrous conditions were performedusing oven-dried glassware under an atmosphere of argon. Brine refers toa saturated solution of sodium chloride. Silica for flash columnchromatography was purchased from Sigma.

Preparation of Previously Described Compounds

Compounds 6c and 11-17 were prepared according to previously describedmethods as summarized in the following table

Structure Number Reference

6c  [6]

15  [7]

16  [8]

17  [9]

18 [10]

19 [11]

20 [12]

21 [13]

22 [14]

23 [15]

24 [16]

Chemical Synthesis of New Compounds

N1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N4-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)succinamidefluorescein Conjugate—1a

25 (7.0 mg, 17 μmol) and fluoresceinisothiocyanate (FITC, 6.7 mg, 17μmol) were dissolved in dimethylformamide (DMF, 1 mL) anddiisopropylethylamine (DIPEA, 8 μL, 48 μmol) was added. The reaction wasstirred for 2 h at room temperature, diluted with MeOH (1 mL) andpurified over reversed-phase HPLC (80% A/20% B to 20% A/80% B over 20min). Fractions containing the desired product by mass spectrometry (MS)were pooled and lyophilized to give the product as a yellow powder (12mg, 16 μmol, 95%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=8.31 (s, 1H), 7.91 (d, J=7.5 Hz, 1H),7.25 (d, J=7.5 Hz, 1H), 7.02 (d, J=8.5 Hz, 2H), 6.92 (s, 2H), 6.79 (d,J=8.5 Hz, 2H), 3.85 (br, 2H), 3.75 (t, J=4.8 Hz, 2H), 3.72-3.65 (m, 4H),3.58 (t, J=5.4 Hz, 2H), 3.38 (t, J=5.4 Hz, 2H), 2.85 (t, J=6.8 Hz, 2H),2.66 (t, J=6.8 Hz, 2H); ¹³C-NMR (125 MHz, DMSO-d₆) δ [ppm]=180.8, 172.3172.2, 171.3, 171.2, 169.0, 164.6, 161.4, 159.9, 152.4, 147.6, 141.8,129.5, 127.0, 124.5, 116.7, 113.0, 110.3, 102.7, 70.1, 69.6, 68.9, 44.1,39.0, 30.7, 29.8, signals from PEG linker predicted to overlap; HRMS.(m/z) [M+H]⁺ calcd. for C₃₃H₃₄N₇O₁₁S₃, 800.1473; found 800.1470.

N1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N4-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)succinamideAlexa546 Conjugate—1 b

To 25 (212 μg, 517 nmol) in DMF (2.1 μL) was added Alexa546 NHS ester(100 rig, 86 nmol). DIPEA (2 μL, 12 μmol) and DMF (50 μL) were added andthe mixture stiffed for 2 h at room temperature. The reaction wasdiluted with MeOH (50 μL) and purified over reversed-phase HPLC (95%A/5% B to 20% A/80% B over 20 min). Fractions containing the product asidentified through its characteristic UV/VIS spectrum (λ_(max)=550 nm)were pooled, lyophilized and dissolved in 100 μL PBS pH 7.4 to give adark purple solution. Its concentration and the reaction yield weredetermined by measuring the absorbance at 556 nm (ε₅₅₆=112,000 M⁻¹ cm⁻¹)of stock samples diluted 1:100 into PBS pH 7.4 (443 μM, 44 nmol, 51%).

HRMS: (nm/z) [M+H]⁺ calcd. for C₅₂H₆₅Cl₃N₉O₁₇S₅, 1352.2162; found1352.2157.

N1-(2-(2-(2-aminoethoxy)ethozy)ethyl)-N4-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)succonamideIRDye750 Conjugate—1c

To 25 (131 μg, 320 nmol) in DMSO (13 μL) was added IRDye750 NHS ester(194 μg, 163 mmol) in DMSO (25 μL) followed by DMF (100 μL) and DIPEA(10 μL, 60 μmol). The solution was stirred for 6 h at room temperatureand then directly purified over reversed-phase HPLC (95% A/5% B to 40%A/60% B over 30 min). Fractions containing dye conjugate were identifiedthrough their characteristic UV/VIS spectrum (λ_(max)=750 nm), pooled,lyophilized and dissolved in dimethylsulfoxide (DMSO, 100 μL) to give adark green stock solution. Its concentration and the reaction yield weredetermined by measuring the absorbance at 750 nm (ε₇₅₀=260,000 M⁻¹ cm⁻¹)of stock samples diluted 1:200 into PBS pH 7.4 (1.02 mM, 102 nmol, 63%).

HRMS: (m/z) [M+Na]²⁻ cacld, for C₆₁H₇₇N₈NaO₁₉S₆, 720.1769; found720.1760.

(S)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-methyl-2-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanamidefluorescein Conjugate—2a

26 (20 mg, 36 μmol) was dissolved in a mixture of dichloromethane (DCM,0.5 mL) and TFA (0.5 mL) and stirred for 1 h at room temperature. Thesolvents were removed under reduced pressure and the residue dissolvedin DMF (0.5 mL). DIPEA (31 μL, 187 μmol) was added followed by FITC (14mg, 36 μmol). The reaction was stirred for 2 h at room temperature,diluted with MeOH (0.5 mL) and purified over reversed-phase HPLC (80%A/20% B to 20% A/80% B over 20 min). Fractions containing product by MSwere pooled end lyophilized to give the product as a bright yellowpowder (18 mg, 23 μmol, 64%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=10.07 (br s, 1H), 8.86 (s, 1H), 8.75(t, J=5.4 Hz, 1H), 8.28 (s, 1H), 8.18 (s, 1H), 8.10 (d, J=6.7 Hz, 2H),7.39 (d, J=6.7 Hz, 2H), 7.74 (d, J=7.9 Hz, 1H), 7.18 (d, J=8.3 Hz, 1H),6.66-6.54 (m, 6H), 5.07 (d, J=10.3 Hz, 1H), 3.68 (br s, 2H), 3.60-3.56(m, 6H), 3.48 (t, J=5.6 Hz, 2H), 3.41-3.19 (m, 2H), 2.49-2.45 (m, 1H),1.01 (d, J=6.6 Hz, 3H), 0.73 (d, J=6.6 Hz, 3H); ¹³C-NMR (125 MHz,DMSO-d₆) δ [ppm]=181.1, 169.0, 167.9, 160.2, 159.1, 158.8, 152.5, 147.3,145.7, 143.6, 141.8. 134.3, 129.5, 126.8, 125.8, 124.6, 121.9, 117.0,114.5, 113.2, 110.3, 102.7, 70.1, 70, 69.6, 69.2, 68.9, 44.2, 31.6,19.2, 19.1; HRMS: (m/z) [M+H]⁺ calcd. for C₄₈₀H₄₂N₇O₁₀₀S₂, 844.2429;found 844.2430.

(S)—N-(2-(2-(2-aminoethoxy)ethozy)ethyl)-3-methyl-2-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanamideIRDye750 Conjugate—2c

26 (178 μg, 321 nmol) in DMSO (34 μL) was added to a mixture of TFA (100μL) and DCM (100 μL). The reaction was stirred for 1 h at roomtemperature and the solvent removed under reduced pressure. To theresidual solution was added IRDye750 NHS ester (194 μg, 163 mmol) inDMSO (25 μL) followed by DMF (100 μL) and DIPEA (10 μL, 60 μmol). Thesolution was stirred for 6 h at room temperature and then directlypurified over reversed-phase HPLC (95% A/5% B to 40% A/60% B over 30min). Fractions containing dye conjugate were identified through theircharacteristic UV/VIS spectrum (λ_(max)=750 mm), pooled, lyophilized anddissolved in DMSO (100 μL) to give a dark green stock solution. Itsconcentration and the reaction yield were determined by measuring theabsorbance at 750 nm (ε₇₅₀₀=260.000 M⁻¹ cm⁻¹) of stock samples diluted1:200 into PBS pH 7.4 (662 μM, 66 nmol, 40%).

HRMS: (m/z) [M+Na]²⁻ calcd. for C₆₈H₈₅N₈NaO₁₈S₅, 742.2247; found742.2233.

4-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)benzenesulfonamidefluorescein Conjugate—3a

28 (7.5 mg, 14 μmol) was dissolved in a mixture of DCM (1 mL) and TFA (1mL) and stirred for 30 min at room temperature. The solvent was removedunder reduced pressure and the residue dissolved in DMF (1 mL). FITC(5.4 mg, 14 μmol) was added followed by DIPEA (23 μL, 139 μmol) and thereaction stirred for 3 h at room temperature. MeOH (1 mL) was added andthe crude reaction mixture purified over reversed-phase HPLC (95% A/5% Bto 20% A/80% B over 20 min). Fractions containing the desired product byMS were pooled and lyophilized to yield the title compound as a brightyellow powder (8.1 mg, 11 μmol, 76%).

¹H-NMR (400 MHz, DMSO-d₆, two rotamers) δ [ppm]=10.31 (br s, 1H), 10.23(br s, 1H), 9.96 (br s, 2H), 8.21 (br s, 2H), 8.02 (br s, 1H), 7.86-7.77(m, 2H), 7.69-7.64 (m, 3H), 7.16 (m, 2H), 7.10 (d, J=8.3 Hz, 1H),6.61-6.48 (m, 6H), 3.61-3.38 (m, 12H); ¹³C-NMR (125 MHz, DMSO-d₆, tworotamers, signals of PEG linker predicted to overlap) δ [ppm]=181.1,169.0, 168.5, 166.0, 160.2, 159.3, 159.0, 157.3, 152.4, 142.5, 141.9,141.0, 139.6, 138.3, 129.5, 127.2, 127.0, 126.9, 124.5, 122.61, 120.0,113.1, 110.5, 102.7, 70.1, 70.0, 69.1, 68.5, 68.9, 49.1, 44.1; HRMS:(m/z) [M+H]⁺ calcd. for C₃₆H₃₄ClN₈O₉S₂, 821.1573; found 821.8567.

4-((4-((2-(2-(2-aminoethoxy)ethoxy)ethyl)amino)-6-chloro-1,3,5-triazin-2-yl)amino)benzenesulfonamideIRDye750 Conjugate—3c

28 (174 μg, 328 nmol) in DMSO (19 μL) was added to a mixture of TFA (100μL) and DCM (100 μL). The reaction was stirred for 1 h at roomtemperature and the volatile solvents removed under reduced pressure. Tothe residual solution, IRDye750 NHS ester (194 μg, 163 mmol) in DMSO (25μL) was added followed by DMF (100 μL) and DIPEA (10 μL, 60 μmol). Thesolution was stirred for 6 h at room temperature and then directlypurified over reversed-phase HPLC (95% A/5% B to 40% A/60% B over 30min).

Fractions containing dye conjugate were identified through theircharacteristic UV/VIS spectrum (λ_(max)=750 nm), pooled, lyophilized anddissolved in DMSO (100 μL) to give a dark green stock solution. Itsconcentration and the reaction yield were determined by measuring theabsorbance at 750 nm (ε₇₅₀₀=260,000 M⁻¹ cm⁻¹) of stock samples diluted1:100 into PBS pH 7.4 (510 μM, 51 nmol, 310).

HRMS: (m/z) IM J; calcd. for C₆₄H₇₇ClN₉O₁₇S₅, 479.4582, found 479.4569.

(E)-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-((4-((4-sulfamoylphenyl)diazenyl)phenyl)amino)propanamideIRDye750 Conjugate—4c

29 (188 μg, 320 nmol) in DMSO (20 μL) was added to a mixture of TFA (100μL) and DCM (100 μL). The reaction was stirred for 1 h at roomtemperature and the volatile solvents removed under reduced pressure. Tothe residue, IRDye750 NHS ester (194 μg, 163 mmol) in DMSO (25 μL) wasadded followed by DMF (100 μL) and DIPEA (10 μL, 60 μmol). The solutionwas stirred for 6 h at room temperature and then directly purified overreversed-phase HPLC (95% A/5% B to 40% A/60% B over 30 min). Fractionscontaining dye conjugate were identified through their characteristicUV/VIS spectrum (λ_(max)=750 nm), pooled, lyophilized and dissolved inDMSO (100 μL) to give a dark green stock solution. Its concentration andthe reaction yield were determined by measuring the absorbance at 750 nm(ε₇₅₀₀=260,000 M⁻¹ cm⁻¹) of stock samples diluted 1:100 into PBS pH 7.4(390 μM, 39 nmol, 24%).

HRMS: (m/z) [M+Na]²⁻ calcd. for C₇₀₀H₈₅N₈NaO₁₈S₅, 754.2247; found754.2248.

N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-sulfamoylbenzamide fluoresceinConjugate-

31 (5.0 mg, 12 μmol) was dissolved in a mixture of DCM (0.5 mL) and TFA(0.5 mL) and stirred for 1 h at room temperature. The solvents wereremoved under reduced pressure and the residue dissolved in DMF (0.5mL). DIPEA (31 μL, 187 μmol) was added followed by FITC (4.5 mg, 12μmol). The reaction was stirred for 2 h at room temperature, dilutedwith MeOH (0.5 mL) and purified over reversed-phase HPLC (80% A/20% B to20% A/80% B over 20 min). Fractions containing product by MS were pooledend lyophilized to give the product as a bright yellow powder (6.3 mg,10 μmol, 83%).

¹H-NMR (400 MHz, DMSO-d₆, only SO₂NH₂ but not NH and OH visible) δ[ppm]=8.74 (t, J=5.2 Hz, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.00 (d, J=8.1Hz, 2H), 7.90 (d, J=8.1 Hz, 2H), 7.74 (d, J=7.9 Hz, 1H), 7.17 (d, J=8.2Hz, 1H), 6.65-6.54 (m, 6H), 3.68 (br s, 2H), 3.61-3.56 (m, 8H),3.47-3.42 (m, 2H); ¹³C-NMR (125 MHz, DMSO-d₆, signals from PEG linkerpredicted to overlap) 5 [ppm]=181.05, 169.0, 165.8, 160.0, 159.1, 158.8,152.3, 152.4, 147.5, 146.7, 141.8, 137.8, 129.5, 128.3, 127.0, 126.1,124.6, 116.9, 113.1, 110.2, 102.7, 70.1, 70.0, 69.3, 68.9, 44.2; HRMS:(m/z) [M+H]⁺ calcd. for C₃₄H₃₃N₄O₁₀₀S₂, 721.1633; found 720.1620.

N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-sulfamoylbenzamide IRDye750Conjugate—5c

31 (138 μg, 320 nmol) in DMSO (29 μL) was added to a mixture of TFA (100μL) and DCM (100 μL). The reaction was stirred for 1 h at roomtemperature and the volatile solvents removed under reduced pressure. Tothe residual solution. IRDye750 NHS ester (194 μg, 163 nnmol) in DMSO(25 μL) was added followed by DMF (100 μL) and DIPEA (10 μL, 60 μmol).The solution was stirred for 6 h at room temperature and then directlypurified over reversed-phase HPLC (95% A/5% B to 40% A/60% B over 30min). Fractions containing dye conjugate were identified through theircharacteristic UV/VS spectrum (λ_(max)=750 nm), pooled, lyophilized anddissolved in DMSO (100 μL) to give a dark green stock solution. Itsconcentration and the reaction yield were determined by measuring theabsorbance at 750 nm (ε₇₅₀₀=260,000 M⁻¹ cm⁻¹) of stock samples diluted1:200 into PBS pH 7.4 (1.23 mM, 123 nmol, 75%).

HRMS: (m/z) [M+2H]²⁺ calcd. for C₆₂H₇₈N₅O₁₈S₅, 1340.3951; found1340.3932.

tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate fluoresceinConjugate—6a

19 (10 mg, 40 μmol) and FITC (16 mg, 41 μmol) were dissolved in DMF (1mL) and DIPEA (10 μL, 48 μmol) was added. The reaction was stirred for 2h at room temperature, diluted with MeOH (1 mL) and purified overreversed-phase HPLC (80% A/20% B to 20% A/80% B over 20 min). Fractionscontaining the desired product by MS were pooled and lyophilized to givethe product as a yellow powder (18 mg, 29 μmol, 72%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=8.45 (br, 1H), 7.76 (br, 1H), 7.18 (d,J=8.3 Hz, 1H), 6.71-6-69 (m, 2H), 6.37-6.56 (m, 4H), 3.63-3.53 (m, 8H),3.39 (t, J=6.0, 2H), 3.07 (br, 2H), 1.35 (s, 9H); ¹³C-NMR (125 MHz,DMSO-d₆) δ [ppm]=180.9, 169.0, 159.9, 156.0, 152.4, 147.4, 141.8, 129.5,129.7, 127.0, 124.5, 116.7, 113.0, 110.3, 110.2, 102.7, 78.1, 70.1,70.0, 69.7, 68.9, 67.02, 44.0, 28.6; HRMS: (m/z) [M+H]⁺ calcd. forC₃₂H₃₆N₃O₉S, 638.2167; found 638.2160.

tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate Alexa546Conjugate—6b

To 19 (233 μg, 943 nmol) in DMSO (6 μL) was added Alexa546 NHS ester(100 μg, 86 nmol). DIPEA (2 μL, 12 μmol) and DMF (50 μL) were added andthe mixture stirred for 2 h at room temperature. The reaction wasdiluted with MeOH (50 μL) and purified over reversed-phase HPLC (95%A/5% B to 20% A/80% B over 20 min). Fractions containing the product asidentified through its characteristic UV/VIS spectrum were pooled(λ_(max)=550 nm), lyophilized and dissolved in 100 μL PBS pH 7.4 to givea dark purple solution. Its concentration and the reaction yield weredetermined by measuring the absorbance at 556 nm (ε₅₅₆=112.000 M⁻¹ cm⁻¹)of stock samples diluted 1:100 into PBS pH 7.4 (555 μM, 56 nmol, 65%).

HRMS: (m/z) [M+2H]⁺ calcd. for C₅₁H₆₇Cl₃N₅O₁S₃, 1190.2856; found1190.2859.

AAZ Targeted CBI Carbonate—7a

AAZ targeted charged linker 11a (7.8 mg, 8.6 μmol) and 12 (5.1 mg, 7.2μmol) were dissolved in degassed MeOH (0.5 mL) and stirred for 6 h atroom temperature. The reaction mixture was directly purified overreversed-phase HPLC (95% A/5% B to 20% A/80% B over 20 min), fractionscontaining the product by MS were pooled and lyophilized to give thetitle compound as a white powder (5.5 mg, 3.7 μmol, 47%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=11.57 (s, 1H), 8.36 (s, 1H), 8.31 (brs, 3H), 8.20 (d, J=7.5 Hz, 1H), 8.06 (d, J=8.5 Hz, 1H), 8.03 (br s, 2H),7.89 (d, J=8.5 Hz, 2H), 7.85 (s, 1H), 7.63 (t, J=8.0, 11H), 7.53 (t,J=8.0 Hz, 1H), 7.31 (s, 1H), 7.21 (br s, 4H), 7.13 (d, J 2.0 Hz, 1H),7.02 (s, 1H), 4.87 (t, J=10.0 Hz, 1H), 4.63-4.52 (m, 5H), 4.45-4.40 (m,1H), 4.29-4.26 (m, 6H), 4.10 (dd, J=11.2, 3.1 Hz, 1H), 4.00 (dd, J=11.2,6.9 Hz, 1H), 3.84 (s, 3H), 3.51-3.49 (m, 2H), 3.25-3.21 (m, 1H),3.14-3.11 (m, 2H), 3.07-3.02 (m, 3H), 2.90 (s, 6H), 2.73-2.57 (m, 6H),2.55-2.45 (m, 2H), 2.13 (t, J=7.1 Hz, 2H), 1.96-1.90 (m, 2H), 1.81-1.73(m, 3H), 1.55-1.40 (m, 5H); HRMS: (m/z) [M+2H]² calcd. forC₆₀₀H₇₈ClN₁₇O₁₉S₄, 751.7110; found 751.7109.

Untargeted CBI Carbonate—7b

Activated carbonate 12 (5.0 mg, 7.1 μmol) and untargeted charged linker11b (10 mg, 14 μmol) were dissolved in degassed MeOH (0.5 mL) andstirred for 6 h at room temperature. The reaction mixture was directlypurified over reversed-phase HPLC (95% A/5% B to 20% A/80% B over 20min), fractions containing the product by MS were pooled and lyophilizedto give the title compound as a white powder (4.1 mg, 3.1 μmol, 43%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=11.57 (s, 1H), 8.37 (s, 1H), 8.21 (d,J=7.4 Hz, 1H), 8.17 (br s, 2H), 8.06 (d, J=8.5 Hz, 1H), 7.96 (d, J=7.3Hz, 1H), 7.89 (d, J=8.5 Hz, 1H), 7.82 (s, 1H), 7.64 (t, J=7.5 Hz, 1H),7.53 (t, J=8.0 Hz, 1H), 7.40-6.77 (br, 4H), 7.31 (s, 1H), 7.13 (d, J=1.9Hz, 1H), 7.03 (s, 1H), 4.86 (t, J=10.1 Hz, 1H), 4.63-4.41 (m, 7H),4.30-4.21 (m, 5H), 4.10 (dd, J=11.1, 2.8 Hz, 1H), 4.0) (dd, J=11.2, 7.0Hz, 1H), 3.85 (s, 3H), 3.54-3.52 (m, 2H), 3.22-2.98 (m, 6H), 2.92 (s,6H), 2.75-2.66 (m, 2H), 2.59 (t, J=7.6 Hz, 2H), 2.54-2.47 (m, 2H), 2.24(t, J=7.4 Hz, 2H), 2.13 (t, J=7.2 Hz, 2H), 1.82-1.70 (m, 5H), 1.53-1.41(m, 5H); HRMS: (m/z) [M+H]⁺ calcd. for C₅₈H₇₅ClN₁₃O₁₈S₂, 1340.4477;found 1340.4466.

AAZ Targeted CBI Carbamate—8a

AAZ targeted charged linker 11a (7.6 mg, 8.3 μmol) and activatedcarbamate 13 (2.7 mg, 3.8 μmol) were dissolved in degassed MeOH (0.5 mL)and stirred for 6 h at room temperature. The reaction mixture wasdirectly purified over reverse-phased HPLC (95% A/5% B to 20% A/80% Bover 20 min), fractions containing the product by MS were pooled andlyophilized to give the title compound as a white powder (2.0 mg, 1.3μmol, 35%).

¹H-NMR (500 MHz, DMSO-d₆, mixture of 2 rotamers) 6 [ppm]=13.00 (br s,1H), 12.39 (br s, 1H), 11.54 (s, 1H), 9.72 (br s, 1H), 8.32 (s, 2H),8.24-8.18 (m, 3H), 8.14-8.10 (m, 1H), 8.03-7.97 (m, 2H), 7.92-7.86 (m,1H), 7.83 (s, 1H), 7.62-7.58 (m, 1H), 7.51-7.44 (m, 2H), 7.32-7.01 (m,7H), 4.85 (t, J=10.6 Hz, 1H), 4.61-4.47 (m, 4H), 4.43-4.38 (m, 1H),4.30-4.19 (m, 5H), 4.11-4.08 (m, 1H), 3.99-3.89 (m, 2H), 3.85 (s, 3H),3.63-3.53 (m, 4H), 3.25-2.96 (m, 8H), 2.93 (s, 6H), 2.75-2.57 (m, 6H),2.53-2.47 (m, 2H), 2.13 (t, J=7.0 Hz, 2H), 1.96-1.90 (m, 2H), 1.78-1.68(m, 3H), 1.54-1.40 (m, 5H); HRMS: (m/z) [M+2H]²⁺ calcd. forC₆₁H₈₁ClN₁₈O₁₈S₄, 758.2268; found 758.2267.

Untargeted CBI Carbamate—8b

Activated carbamate 13 (5.0 mg, 6.9 μmol) and untargeted charged linker11b (10 mg, 14 μmol) were dissolved in degassed MeOH (0.5 ml.) andstirred for 6 h at room temperature. The reaction mixture was directlypurified over reverse-phased HPLC (95% A/5% B to 20% A/80% B over 20min), fractions containing the product by MS were pooled and lyophilizedto give the title compound as a white powder (5.1 mg, 3.7 μmol, 54%).

¹H-NMR (500 MHz, DMSO-d₅, mixture of 2 rotamers) δ [ppm]=12.4 (br s, 3H)11.54 (s, 1H), 8.24-8.14 (m, 4H), 8.03-7.87 (m, 3H), 7.82 (s, 1H),7.63-7.47 (m, 3H), 7.32-6.82 (m, 7H), 4.85 (t, J=10.0 Hz, 1H), 4.624.53(m, 3H), 4.50-4.44 (br m, 1H), 4.43-4.37 (br m, 1H), 4.30-4.20 (m, 5H),4.11-4.07 (m, 1H), 3.99-3.87 (m, 2H), 3.85 (s, 3H), 3.64-3.50 (m, 4H),3.25-2.95 (m, 8H), 2.93 (s, 6H), 2.75-2.58 (m, 4H), 2.53-2.46 (m, 2H),2.25 (t, J=7.4 Hz, 2H), 2.12 (t, J=7.3 Hz, 2H), 1.84-1.68 (m, 5H),1.56-1.38 (m, 5H); HRMS: (m/z) [M+2H]²⁺ calcd. for C₅₉H₇₉ClN₁₄O₁₇S₂,677.2433; found 677.2430.

Targeted DM1 Conjugate—9a

CysAspArgAsp(SEQ ID NO: 1)-Linker-AAZ 11a (40 mg, 40 μmol) was dissolvedin degassed MeOH (5 mL) and 2,2′-dipyridyldisulfide (13.2 mg, 60 μmol)was added. The mixture was stirred at room temperature for 12 h andadded drop wise to ice cold diethyl ether (40 mL). The precipitate wascollected by centrifugation, re-dissolved in MeOH and precipitated againwith ice cold diethyl ether (40 mL) and dried under vacuum to give theactivated disulfide as a white residue (20 mg, 20 μmol, 49%). An aliquotof the activated disulfide (8 mg, 7.8 μmol) was dissolved in DMF (500μL) and DM1 free thiol (5.5 mg, 7.4 μmol) added. The reaction wasallowed to stand at room temperature for 48 h after which the productwas recovered by reversed phase HPLC (95% A/5% B to 20% A/80% B over 20min). Fractions containing the desired product by MS were pooled andlyophilized to yield the title compound as an off white powder (9.0 mg,5.5 μmol, 74%).

HRMS: (m/z) [M+2H]²⁺ calcd. for C₆₅H₉₄ClN₁₇O₂₃S₄ 821.7634; found821.7633.

Untargeted DM1 Conjugate—9b

CysAspArgAsp-Linker-COOH (SEQ ID NO: 1) 11b (21 mg, 28 μmol) wasdissolved m degassed MeOH (5 mL) and reduced with TCEP HCl (16 mg, 56μmol) for 2 h at room temperature, 2,2′-Dipyridyldisulfide (25 mg, 114μmol) was added and the mixture stirred for 12 h at room temperature.The reaction was precipitated into ice cold diethyl ether (40 mL), theproduct collected by centrifugation, re-dissolved in MeOH (5 mL) andprecipitated again with ice cold diethyl ether (40 mL). The precipitatewas dried under vacuum to give the activated disulfide as a whiteresidue (20 mg, 23 μmol, 83%). An aliquot of the activated disulfide (10mg, 12 μmol) was dissolved in DMF (500 μL) and DM1 free thiol (8.6 mg,12 μmol) was added. The reaction was allowed to stand at roomtemperature for 48 h after which the product was recovered by reversedphase HPLC (95% A/5% B to 20% A/80% B over 20 min). Fractions containingthe desired product by MS were pooled and lyophilized to yield the titlecompound as an off white powder (7.0 mg, 4.7 μmol, 40%).

HRMS: (m/z) [M+2H]²⁺ calcd. for C₆₃H₉₂ClN₁₃O₂₂S₂, 740.7799; found740.7792.

N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)hex-5-ynamide—10

A solution of 5-hexynoic acid (1.4 mL, 12.9 mmol) and DMF (50 μL) in DCM(50 mL) was cooled on ice and oxalyl chloride (1 mL, 11.7 mmol) wasadded drop wise over 15 min. The reaction was allowed to warm to roomtemperature, stirred until evanescence ceased and then concentratedunder reduced pressure. The yellow liquid was added drop wise to asolution of 23 (2.3 g, 12.9 mmol) and pyridine (943 μL, 25.8 mmol) inDMF (15 mL) and the reaction stirred for 3 h at room temperature. Thesolvent was removed under reduced pressure and the residue purified byflash column chromatography (EtOAc) to give the product as an off-whitesolid (2.8 g, 79%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=2.81 (t, J=2.6 Hz, 1H), 2.65 (t, J=7.4Hz, 2H), 2.24 (td, J=7.1, 2.6 Hz, 2H), 1.84-1.77 (m, 2H); ¹³C-NMR (100MHz, DMSO-d₆) δ [ppm]=171.5, 164.2, 160.9, 83.6, 71.8, 33.6, 23.1, 17.2;HRMS: (m/z) [M+H]⁺ calcd. for C₈H₁₁N₄O₃S₆, 275.0267; found 275.0268.

CysAspArgAsp-Linker-Acetazolamide—11a (SEQ ID NO: 1)

Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500mg, 0.415 mmol, RAPP Polymere) was swollen in DMF (3×5 min×5 mL), theFmoc group removed with 20% piperidine in DMF (1×1 min×5 mL and 2×10min×5 mL) and the resin washed with DMF (6×1 min×5 mL). The peptide wasextended with Fmoc-Asp(tBu)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Asp(tBu)-OH inthe indicated order and then capped with 5-azido-valerate. For thispurpose, the Fmoc protected amino acid or azido acid (3.0 eq), HBTU (3.0eq), HOBt (3.0 eq) and DIPEA (6.0 eq) were dissolved in DMF (5 mL), themixture was allowed to stand for 1 min at room temperature and thenreacted with the resin for 1 h under gentle agitation. After washingwith DMF (6×1 min×5 mL) the Fmoc group was removed with 20% piperidinein DMF (1×1 min×5 min and 2×10 min×5 mL) and the resin washed with DMF(6×1 min×5 mL) before the next coupling step was initiated. Aftercoupling of 5-azido-valerate, a solution of CuI (0.3 eq), TBTA (0.3 eq)and alkyne 10 (6 eq) in a mixture of DMF (2.5 mL) and THF (2.5 mL) wasprepared and reacted with the resin for 2 h at room temperature. Afterwashing with DMF (3×1 min×5 mL), 50 mM aq. EDTA solution (3-1 min×5 mL),DMF (3×1 min×5 mL) and DCM (3×1 min×5 mL), the compound was cleaved byagitating the resin with a mixture of TFA (4.5 mL), TIS (250 μL) and H₂O(250 μL) for 2 h at room temperature. The resin was washed with TFA (1×5min×5 mL) and the combined cleavage and washing solutions addeddrop-wise to ice cold diethyl ether (100 mL). The precipitate wascollected by centrifugation and the product purified by reversed-phaseHPLC (95% A/5% B to 20% A/80% B over 20 min). After lyophilization thetitle compound was collected as a white powder (135 mg, 0.14 mmol, 33%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=13.00 (s, 1H), 8.31 (s, 2H), 8.23-8.20(m, 2H), 7.98 (d, J=7.5 Hz, 1H), 7.93 (d, J=7.8 Hz, 1H), 7.82 (br m,1H), 7.53 (br m, 1H), 7.27-7.06 (br m, 4H), 4.59-4.51 (m, 2H), 4.40-4.36(m, 1H), 4.27 (t, J=6.7 Hz, 2H), 4.21-4.20 (m, 1H), 3.06-3.04 (br m,2H), 2.87-2.47 (m, 9H), 2.38 (t, J=8.6 Hz, 1H), 2.14 (t, J=7.0 Hz, 2H),1.95-1.90 (m, 2H), 1.77-1.69 (m, 3H), 1.54-1.39 (m, 5H); ¹³C-NMR (125MHz, DMSO-d₆) δ [ppm]=172.7, 172.5, 172.3, 172.1, 171.7, 171.6, 171.5,170.9, 164.7, 161.5, 157.2, 146.5, 122.3, 54.9, 52.7, 50.1, 49.9, 49.3,40.9, 36.3, 36.2, 34.7, 29.6, 29.4, 35.9, 25.2, 24.8, 24.6, 22.5; HRMS:(m/z) [M+H]⁴ calcd. for C₃₀₀H₄₇N₁₄O₁₃S₃, 995.1966; found 995.1964.

CysAspArgAsp-Linker-COOH—11b

Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500mg, 0.415 mmol, RAPP polymere) was swollen in DMF (3×5 min×5 mL), theFmoc group removed with 20% piperidine in DMF (1×1 min×5 mL and 2×10min×5 mL) and the resin washed with DMF (6×1 min×5 mL). The peptide wasextended with Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH and Fmpc-Asp(OtBu)-OHin the indicated order and then capped with 5-azido-valerate. For thispurpose, the Fmoc protected amino acid or azido acid (3.0 eq), HBTU (3.0eq), HOBt (3.0 eq) and DIPEA (6.0 eq) were dissolved in DMF (5 mL), themixture was allowed to stand for 1 min at room temperature and thenreacted with the resin for 1 h under gentle agitation. After washingwith DMF (6×1 min×5 mL) the Fmoc group was removed with 20% piperidinein DMF (1×1 min×5 min and 2×10 min×5 mL) and the resin washed with DMF(6×1 min×5 mL) before the next coupling step was initiated. Aftercoupling of 5-azido-valerate, a solution of CuI (0.3 eq), TBTA (0.3 eq)and 5-hexynoic acid (6 eq) in a mixture of DMF (2.5 mL) and THF (2.5 mL)was prepared and reacted with the resin for 2 h at room temperature.After washing with DMF (3×1 min×5 mL), 50 mM aq. EDTA solution (3×1min×5 mL), DMF (3×1 min×5 mL) and DCM (3×1 min×5 mL), the compound wascleaved by agitating the resin with a mixture of TFA (4.4 mL), phenol(250 μL), water (250 μL) and TIPS (100 μL) for 2 h at room temperature.The resin was washed with TFA (1×5 min×5 mL) and the combined cleavageand washing solutions added drop-wise to ice cold diethyl ether (100mL). The precipitate was collected by centrifugation and the productpurified by reversed-phase HPLC (95% A/5% B to 20% A/80% B over 20 min).After lyophilization the title compound was collected as a white powder(116 mg, 0.16 mmol, 43%).

¹H-NMR (500 MHz, MeOH-d₄) δ [ppm]=7.81 (s, 1H), 4.58-4.51 (m, 2H),4.30-4.23 (m, 4H), 3.09 (t, J=6.9 Hz, 2H), 2.91-2.60 (m, 8H), 2.25 (t,J=7.4 Hz, 2H), 2.18 (t, J=7.4 Hz, 2H), 1.90-1.77 (m, 5H), 1.65-1.48 (m,5H); ¹³C-NMR (125 MHz, DMSO-d₆) δ [ppm]=174.6, 173.6, 172.7, 172.2,172.0, 171.6, 171.4, 170.7, 157.1, 146.7, 122.3, 54.8, 52.7, 49.9, 49.8,49.3, 40.8, 36.2, 34.7, 33.4, 33.1, 29.6, 25.8, 25.1, 24.8, 24.7, 22.5;HRMS: (m/z) [M+H]⁺ calcd. for C₂₈H₄₆N₁₀₀O₁₂S, 745.2934; found 745.2931.

Seco CBI Drug carbonate pyridyl disulfide—12

Seco CBI drug 14 (10 mg, 20 μmol, 1 eq), activated carbonate 16 (7.2 mg,20 μmol, 1 eq) and N,N-dimethylaminopyridine (DMAP, 2.4 mg, 50 μmol, 2.5eq) were dissolved in DMF (2 mL) and stirred for 5 h at roomtemperature. The reaction mixture was diluted with MeOH (2 mL) andpurified over HPLC (95% A/5% B to 20% A/80% B over 20 min). Fractionscontaining the desired product my MS were pooled and lyophilized toyield the title compound as an off white powder (10.1 mg, 14.3 μmol,72%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=8.45 (br s, 1H) 8.34 (s, 1H),7.94-7.81 (m, 4H), 7.66 (dt, J=6.9, 1.1 Hz, 1H), 7.59 (dt, J=6.9, 1.0Hz, 1H), 7.33 (s, 1H), 7.26 (m, 1H), 7.06 (s, 1H), 6.99 (s, 1H),4.70-4.61 (m, 2H), 4.58 (t, J=6.0 Hz, 2H), 4.33 (t, J=4.9 Hz, 2H),4.24-4.20 (m, 1H), 4.00 (dd, J=11.4, 3.3 Hz, 1H), 3.89 (s, 3H), 3.71(dd, J=11.3, 8.2 Hz, 1H), 3.60 (t, J=4.9 Hz, 2H), 3.25 (t, J=6.0 Hz,2H), 3.06 (s, 6H); ¹³C-NMR (125 MHz, MeOD-d₄) δ [ppm]=160.3, 159.3,153.8, 150.0, 149.2, 146.9, 143.4, 141.2, 137.9, 132.9, 129.7, 128.7,127.5, 125.1, 123.8, 122.9, 122.7, 121.6, 121.4, 120.6, 120.0, 110.6,107.8, 106.7, 93.8, 66.3, 64.5, 56.6, 54.8, 46.3, 42.5, 42.0, 37.0.HRMS: (m/z) [M+H]⁺ calcd. for C₃₅H₃₆ClN₄O₆S₂, 707.1759; found 707.1761.

Seco CBI Drug carbamate pyridyl disulfide—13

Seco CBI drug 14 (10 mg, 20 μmol, 1.0 eq), activated carbamate 17 (12mg, 92 μmol, 4.6 eq) and DMAP (10 mg, 100 μmol, 5.0 eq) were dissolvedin DMF (2 mL) and stirred for 12 h at room temperature. The reactionmixture was diluted with MeOH (2 mL) and purified over HPLC (95% A/5% Bto 20% A/80% B over 20 min). Fractions containing the desired product myMS were pooled and lyophilized to yield the title compound as an offwhite powder (9.5 mg, 13 μmol, 65%).

¹H-NMR (400 MHz, MeOD-d₄, mixture of two rotamers) δ [ppm]=8.42-8.23 (brm, 2H), 7.96-7.73 (m, 4H), 7.60-7.37 (m, 2H), 7.32 (s, 1H), 7.35-7.24(m, 1H), 7.16-7.14 (m, 1H), 7.04-7.03 (2s, 1H), 4.74-4.65 (m, 2H), 4.31(t, J=4.7 Hz, 2H), 4.30-4.21 (m, 1H), 4.07-4.00 (m, 2H), 3.84-3.82 (2s,3H), 3.87-3.62 (m, 2H), 3.60 (t, J=4.9 Hz, 2H), 3.36-3.11 (m, 5H), 3.05(s, 6H); ¹³C-NMR (125 MHz, MeOD-d₄) δ [ppm]=161.8, 161.7, 161.1, 160.8,157.0 156.6, 151.5, 150.6, 149.1, 144.9, 142.9, 142.8, 139.1, 134.6,131.3, 128.8, 126.3, 124.1, 123.8, 123.7, 123.6, 122.6, 122.2, 121.5,121.5, 112.9, 112.6, 109.5, 108.0, 107.9, 95.5, 98.4, 66.2, 58.1, 56.4,56.3, 47.8, 44.0, 43.6, 37.5, 37.4, 35.8, 35.7; HRMS: (m/z) [M+H]⁺calcd. for C₃₆H₃₉ClN₅O₅S₂, 720.2076; found 720.2074.

Seco CBI Drug—14

N-Boc protected seco CBI (50 mg, 150 μmol, 1.0 eq) was dissolved in 4 MHCl in dry EtOAc (5 mL) and stirred for 6 h at room temperature. Thesolvent was removed under reduced pressure and the residue dissolved in3 mL DMF and cooled on ice. EDC HCl (86 mg, 450 μmol, 3.0 eq) was addedfollowed by indole 18 (61 mg, 220 μmol, 1.3 eq), the mixture warmed toroom temperature and allowed to stir for 12 h. MeOH (3 mL) was added andthe crude reaction mixture purified over HPLC (95% A/5% B to 20% A/80% Bover 20 min). Fractions containing the desired product my MS were pooledand lyophilized to yield the title compound as an off-yellow powder(44.1 mg, 89.5 μmol, 60%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=8.10 (d, J=8.2 Hz, 1H), 7.77 (s, 1H),7.61 (d, J=8.3 Hz, 1H), 7.37 (td, J=6.8, 1.2 Hz, 1H), 7.23 (td, J=6.8,1.0 Hz, 1H), 7.15 (s, 1H), 6.91 (s, 1H), 6.87 (s, 1H), 4.49-4.38 (m,2H), 4.13 (t, J=4.8 Hz, 2H), 3.96-3.92 (m, 1H), 3.80 (dd, J=11.2, 3.2Hz, 1H), 3.72 (s, 3H), 3.44-3.39 (m, 3H), 2.91 (s, 6H); ¹³C-NMR (100MHz, MeOD-d₄) δ [ppm]=173.0, 162.4, 155.8, 151.6, 144.9, 143.4, 134.6,131.6, 130.9, 128.6, 124.6, 124.5, 123.5, 122.2, 117.0, 109.5, 107.9,101.5, 95.5, 66.0, 58.1, 56.8, 56.3, 47.5, 43.9, 43.6; HRMS: (m/z)[M+H]⁺ calcd. for C₂₇H₂₉ClN₃O₄, 494.1841; found 494.1843.

N1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-N4-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)succinamide—25

Commercially available pre-loaded O-Bis-(aminoethyl)ethylene glycol ontrityl resin (500 mg, 0.3 mmol, Merck Millipore) was swollen in DMF (3×5min×5 mL), 4-oxo-4-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino)butanoicacid (AAZSucc, 166 mg, 0.59 mmol) and HATU (228 mg, 0.60 mmol) weredissolved in DMF (5 mL) and DIPEA (200 μL, 1.2 mmol) was added. Thesolution was immediately reacted with the resin for 30 mm at roomtemperature. The resin was washed with DMF (6×1 min×5 mL), DCM (3×1min×5 mL) and cleaved with 95% TFA/2.5% H₂O/2.5% triisopropylsilane(TIS, 5 mL total volume) for 1 h at room temperature and washed with TFA(1×1 min×5 mL). The combined cleavage and wash solutions were pouredinto cold Et₂O (40 mL), the precipitate collected by centrifugation andpurified over reversed-phase HPLC (95% A/5% B to 20% A/80% B over 20min). Fractions containing the desired product by MS were pooled andlyophilized to give the product as a white powder (48 mg, 0.12 mmol,39%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=3.60 (t, J=5.0 Hz, 2H), 3.55 (m, 4H),3.45 (t, J=5.6 Hz, 2H), 3.27 (t, J=5.6 Hz, 2H), 3.02 (t, J=5.0 Hz, 2H),2.75 (t, J=5.6 Hz, 2H), 2.54 (t, J=6.2 Hz, 2H); ¹³C-NMR (100 MHz,MeOD-d₄) δ [ppm]=174.3, 173.0, 166.4, 163.1, 71.4, 71.3, 70.7, 67.9,40.7, 40.3, 31.5, 30.9; HRMS: (m/z) [M+H]⁺ calcd. for C₁₂H₂₃N₆O₆S₂,411.1115; found 411.1116.

(S)-tert-butyl(2-(2-(2-(3-methyl-2-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanamido)ethoxy)ethoxy)ethyl)carbamate—26

To a solution of 27 (64 mg, 0.20 mmol) in DMF (2 mL) was added NHS (25mg, 0.22 mmol) and EDC·HCl (42 mg, 0.22 mmol) and the mixture wasstirred for 1 h at room temperature. A solution of 19 (54 mg, 0.22 mmol)and DIPEA (110 μL, 0.67 mmol) in DMF (1 mL) was added and the reactionstirred for 1 h at room temperature. The solvent was removed underreduced pressure, the residue dissolved in DCM (5 mL) and the solutionwashed with H₂O (1×5 mL), brine (1×5 mL), dried over Na₂SO₄ and thesolvent removed under reduced pressure. Purification by flash columnchromatography over silica (EtOAc) gave the product as a white solid (63mg, 0.11 mmol, 57%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=8.75 (br m, 1H), 8.69 (s, 1H), 8.05(d, J=6.6 Hz, 2H), 7.98 (d, J=6.6 Hz, 2H), 3.61-3.56 (m, 6H), 3.53-3.37(m, 4H), 3.22 (t, J=5.6 Hz, 2H), 2.65-2.56 (m, 1H), 1.44 (s, 9H), 1.12(d, J=6.7 Hz, 3H), 0.84 (d, J=6.6 Hz, 3H); ¹³C-NMR (100 MHz, MeOD-d₄) δ[ppm]=170.0, 158.5, 147.5, 144.5, 135.5, 128.0, 127.0, 122.3, 80.1,71.7, 71.6, 71.3, 71.1, 70.3, 41.2, 40.7, 33.0, 28.8, 19.6, 19.2; HRMS:(m/z) [M+H]⁺ calcd. for C₂₄H₃₈N₆NaO₇S, 577.2415; meas. 577.2415.

(S)-3-methyl-2-(4-(4-sulfamoylphenyl)-1H-1,2,3-triazol-1-yl)butanoicacid—27

Ethynyl benzene sulfonamide (54 mg, 0.3 mmol), azido valine 20 (42 mg,0.3 mmol) and tris-(benzyltriazolylmethyl)amine (TBTA, 0.3 mg, cat.)were dissolved in a mixture of tBuOH (3.7 mL), a 0.04 M CuSO₄ solutionin PBS pH 7.4 (2.0 mL) and a 0.1 M sodium ascorbate solution in PBS pH7.4 (1.7 mL) and stirred for 12 h at room temperature. All solvents hadpreviously been de-gassed and flushed with Ar. The reaction was pouredonto 25 mL H₂O acidified to pH 2.0 and the mixture extracted with EtOAc(4×20 mL), the pooled organic phases washed with brine and dried overMgSO₄. The solvent was removed under vacuum and the residue purifiedover silica (20% MeOH in DCM with 0.1% Et₃N) to yield the product as awhite solid (70 mg, 72%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=8.86 (s, 1H), 8.10 (d, J=8.5 Hz, 2H),7.90 (d, J=8.5 Hz, 2H), 7.39 (s, 1H), 5.24 (d, J=8.0 Hz, 1H), 2.64-2.55(m, 1H), 1.00 (d, J=6.7 Hz, 3H), 0.88 (d, J=6.7 Hz); ¹³C-NMR (100 MHz,DMSO-d₆) 5 [ppm]=169.6, 145.0, 143.2, 133.7, 126.3, 125.4, 122.6, 68.3,30.4, 19.0, 18.3; HRMS: (m/r) [M+H]⁺ calcd. for C₁₃H₁₅N₄Na₂O₄S,369.0604; found 369.0609.

tert-butyl(2-(2-(2-((4-chloro-6-((4-sulfamoylphenyl)amino)-1,3,5-triazin-2-yl)amino)ethoxy)ethoxy)ethyl)carbamate—28

21 (160 mg, 0.64 mmol), 14 (204 mg, 0.64 mmol) and DIPEA (105 μL, 0.64mmol) were dissolved in DMF (5 mL) and stirred for 3 h at roomtemperature. The reaction mixture was diluted with H₂O (15 mL) andextracted with EtOAc (3×10 mL). The combined organic fractions werewashed with 10% w/v aq. LiCl solution (1×10 mL), brine (1×10 mL) anddried over Na₂SO₄. The solvent was removed under reduced pressure andthe residue purified over silica (EtOAc) to give the product as a whitesolid (239 mg, 66%).

¹H-NMR (400 MHz, DMSO-d₆, mixture of two rotamers) δ [ppm]=10.39-10.30(m, 1H), 8.27 (br s, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.85 (d, J=8.1 Hz,1H), 7.74 (m, 2H), 7.25 (s, 1H), 7.23 (s, 1H), 6.74-6.73 (m, 1H),3.57-3.32 (m, 10H), 3.05 (m, 2H), 1.37 (s, 9H); ¹³C-NMR (125 MHz,DMSO-d₆, mixture of two rotamers, several signals overlap) δ[ppm]=169.0, 168.5, 166.0, 165.9, 164.2, 163.7, 156.0, 142.6, 142.5,138.3, 138.2, 127.1, 126.8, 120.0, 119.8, 78.1, 70.1, 70.0, 69.9, 69.6,69.1, 68.8, 67.1, 39.0, 28.7; HRMS: (m/z) [M+H]⁺ calcd. forC₂₀₀H₃₀₀ClN₇NaO₆S, 554.1559; found 554.1555.

(E)-tert-butyl(2-(2-(2-(3-((4-((4-sulfamoylphenyl)diazenyl)phenyl)amino)propanamido)ethoxy)ethoxy)ethyl)carbamate-29

To 30 (20 mg, 57 μmol) dissolved in DMF (1 mL) was added HOBt (8.7 mg,57 μmol) followed by EDC HCl (12.2 mg, 64 μmol). After stirring thereaction for 1 h at room temperature a solution of 19 (15.8 mg, 64 mmol)and DIPEA (20 μL, 122 μmol) in DMF (0.5 mL) was added. The mixture wasstirred for 1 h at room temperature, diluted with MeOH (1.5 mL) andpurified over reversed-phase HPLC (80% A/20% B to 20% A/80% B over 20min). Fractions containing the desired product by MS were pooled andlyophilized to give the product as an orange powder (26 mg, 45 mmol,79%).

¹H-NMR (400 MHz, MeOD-d₄) δ [ppm]=8.02 (d, J=8.8 Hz, 2H), 7.91 (d, J=8.8Hz, 2H), 7.83 (d, J=9.0 Hz, 2H), 7.76 (d, J=9 Hz, 2H), 3.60 (s, 4H),3.58-3.49 (m, 6H), 3.40 (t, J=5.5 Hz, 2H), 3.23-3.20 (m, 2H), 2.56 (t,J=6.7 Hz, 2H), 1.45 (s, 9H); ¹³C-NMR (125 MHz, MeOD-d₄) δ [ppm]=170.8,156.1, 154.7, 153.2, 144.3, 143.3, 127.4, 126.3, 122.4, 112.2, 78.1,70.0, 69.9, 69.6, 69.5, 39.1, 35.4, 28.7; HRMS: (m/z) [M+H]⁺ calcd. forC₂₆H₃₈N₆NaO₇S, 601.2415; found 601.2416.

(E)-3-((4-((4-sulfamoylphenyl)diazenyl)phenyl)amino)propanoic acid-30

Sulfanilamide (85 mg, 0.49 mmol) was dissolved in 40% aq. HCl (1.3 mL)and cooled to 0° C. A solution of NaNO₂ in water (300 μL) was addeddrop-wise over 5 min and the reaction stirred on ice for 15 min. Theyellowish solution was slowly added to a suspension of 22 (129 mg, 0.37mmol) in 10M aq. NaOH (1 mL) and DMF (1 mL) and stirred for 2 h at roomtemperature. The dark red solution was acidified with 6 N HCl, extractedwith EtOAc (6×10 mL), dried over Na₂SO₄ and the solvent removed underreduced pressure. Recrystallization of the dark red residue gave theproduct as a red solid (32 mg, 25%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=7.95 (d, J=8.6 Hz, 2H), 7.88 (d, J=8.6Hz, 2H), 7.77 (d, J=8.9 Hz, 2H), 7.44 (s, 2H), 9.94 (br s, 1H), 6.74 (d,J=8.9 Hz, 2H), 3.40 (t, J=6.4 Hz, 2H), 2.56 (t, J=6.4 Hz, 2H); ¹³C-NMR(100 MHz, DMSO-d₆) δ [ppm]=172.9, 154.1, 152.6, 143.8, 142.9, 126.8,125.8, 121.9, 111.7, 38.4, 33.4; HRMS: (m/z) [M+H]⁺ calcd. forC_(1S)H₁₇N₄O₄S, 349.0965; found 349.0967.

tert-butyl(2-(2-(2-(4-sulfamoylbenzamido)ethoxy)ethoxy)ethyl)carbamate-31

To a solution of 4-carboxybenzenesulfonamide (46 mg, 0.23 mmol) in MeCN(2 mL) was added NHS (29 mg, 0.25 mmol) followed by EDC HCl (48 mg, 0.25mmol). After stirring for 4 h at room temperature more EDC HCl (24 mg,0.13 mmol) was added and the reaction stirred for a further 1 h at roomtemperature. A solution of 19 (52 mg, 0.21 mmol) and DIPEA (140 μL, 0.85mmol) in DMF (1 mL) was added. After stirring for 10 h at roomtemperature, the reaction was filtered through a pad of silica elutingwith EtOAc, the solvent removed under reduced pressure and the residuepurified over silica (EtOAc to 10% MeOH in EtOAc) to give the product asa white solid (61 mg, 0.14 mmol, 67%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=8.71 (br m, 1H), 8.00 (d, J=8.1 Hz,2H), 7.90 (d, J=8.1 Hz, 2H), 7.46 (br s, 2H), 6.76 (br m, 1H), 3.55-3.33(m, 10H), 3.06-3.05 (m, 2H), 1.37 (s, 9H); ¹³C-NMR (125 MHz, DMSO-d₆) δ[ppm]=165.8, 156.1, 146.7, 137.7, 128.3, 126.1, 78.1, 70.0, 69.9, 69.6,69.2, 67.1, 39.1, 28.7; HRMS: (m/z) [M+H]⁺ calcd. for C₁₈H₂₉N₃NaO₇S,454.1618; found 454.1623.

Propagation of Errors

During data analysis standard deviations were propagated according toformula (1) as recommended by the National Institute of Standards andTechnology.^([2])

$\begin{matrix}{\sigma_{f} = \sqrt{{\sum}_{i = {1\ldots n}}\left( {\frac{\partial f}{\partial x_{i}}\sigma_{i}} \right)^{2}}} & (1)\end{matrix}$

Where ƒ=ƒ(x₁, x₂, . . . x_(n)), σ_(ƒ) is the standard deviation offunction ƒ and σ_(i) is the standard deviation of x_(i).

Determination of Ligand K_(D) by Fluorescence Polarization Measurement

Fluorescently labeled ligands (2 mg) were dissolved in DMSO (100 μL) anddiluted 1:2000 into PBS pH 7.4 to determine the stock's concentration byabsorbance measurement at 495 nm (ε₄₉₅=72,000 M⁻¹ cm⁻¹). RecombinantCAIX was expressed as described previously, dialyzed against assaybuffer (50 mM tris(hydroxymethyl)aminomethane [TIRS] pH 7.4 containing 1mM ZnSO₄) at 4° C. overnight and the protein concentration determined byabsorbance measurement at 280 nm (ε₂₈

=35.075 M⁻¹ cm⁻¹).

In a black 384-well plate in assay buffer (30 μL) fluorescently labeledligands (5 nM from appropriately diluted DMSO stocks, final DMSO contentadjusted to 0.001%) were incubated with increasing concentrations ofrecombinant carbonic anhydrase IX (4.6 μM to 140 μM in steps of 1:2) for1 h at room temperature. The fluorescence polarization (FP) was measuredon a Spectra Max Paradigm multimode plate reader (Molecular Devices).Experiments were performed in triplicate, mean FP values divided by thetop-plateau signal and the fractional FP value fit to equation (2) usingKaleidaGraph 4.0 (Synergy Software).

FP=([P] ₀ +[L] ₀ +K _(D))−√{square root over (([P] ₀ +[L] ₀ +K_(D))²−4[P] ₀ [L] ₀)}  (2)

Where FP is the fractional fluorescence polarization, [P]

the total protein concentration, [L]

the total concentration of the fluorescently labeled ligand and K

the dissociation constant in nM.

Measured values of K_(D) for fluorophore-linker-ligand complexes were:1a 12.6±1.0 nM; 2a 18.1±1.3 nM; 3a 46.8±1.2 nM; 5a 218±9 nM. 4a couldnot be determined due to its dark quenching properties. Referenceexample 6a did not bind to the CAIX. Thus, the acetazolamide (AAZ)ligand of 1a appears to be the most promising.

Competitive Fluorescence Polarization Measurement of K_(D)

In a black 384-well plate in assay buffer (see above, 40 μL)fluorescently labeled probe 1a (5 nM from appropriately diluted DMSOstocks) and recombinant carbonic anhydrase IX (25 nM) were incubatedwith increasing concentrations of unlabeled ligand (2.5 μM to 76 μM insteps of 1:2) for 1 h at room temperature. The FP was measured on aSpectra Max Paradigm multimode plate reader (Molecular Devices).Experiments were performed in triplicate and data analyzed as describedby Wang and co-workers.^([4])

This method was used to determine K_(D) for the unlabeleddrug-linker-ligand conjugates prepared as shown in FIG. 2 . K

values are given in brackets±standard errors of fit. The followingvalues of K_(D) were determined: 7a 7.3±0.5 nM; 8a 40.3±2.6 nM; 9a26.5±2.5 nM. The K

for 7b was >1 μM. Thus, targeted conjugates 7a, 8a and 9a retain bindingaffinity for recombinant CAIX in vitro whereas untargeted controls 7b,8b and 9b do not exhibit strong binding

Cell Culture

SKRC52 and HEK cells were maintained in RPMI medium (Invitrogen)supplemented with 10% fetal calf serum (FCS. Invitrogen) andantibiotic-antimycotic (AA, Invitrogen) at 37° C. and 5% CO₂. A549 cellswere maintained in F-12K medium (Invitrogen) supplemented with 10% FCS(Invitrogen) and AA (Invitrogen) at 37° C. and 5% CO₂. For passaging,cells were detached using trypsin with ethylenediaminetetraacetic acid(EDTA) 0.05% (Invitrogen) when reaching 90% confluence and re-seeded ata dilution of 1:10.

Cell surface expression of CAIX on different cell lines used in thisstudy were analyzed by flow cytometry using aCAIX (a Santa Cruzbiotechnology polyclonal rabbit anti human CAIX antibody). A suitablylabeled anti rabbit IgG antibody was used for detection. It was foundthat SKRC52 cells constitutively express high levels of CAIX. Incontrast, A549 cells express only very low levels of CAIX under normoxicconditions. Since they maintain strong attachments to culture plates, weused these cells as negative controls in experiments requiring multiplewashing steps of attached cells. It was found that HEK cells do notexpress detectable levels of CAIX under normoxic culture conditions.They can easily be detached from culture plates with EDTA and were thusused as negative controls in most flow cytometry experiments.

In Vitro Cytotoxicity Assay

SKRC52 or A549 cells were seeded in 96-well plates in their appropriateculture medium (100 μL) at a density of 5000 cells per well and allowedto grow for 24 h. The medium was replaced with medium containingdifferent concentrations of test substance (100 μL, 300 nM-15 μM in 1:3dilution steps) and plates were either:

-   -   (a) incubated for 72 hours in the presence of the toxic        substance, or    -   (b) incubated for 1 h under standard culture conditions,        followed by removal of the medium containing the toxic        substance, gently washing the cells fresh medium once and adding        new medium (100 μL) and incubating for 72 h under culture        conditions.

MTS cell viability dye (20 μL, Promega) was added, the plates wereincubated for 1 h under culture conditions and the absorbance at 490 nmmeasured on a Spectra Max Paradigm multimode plate reader (MolecularDevices). Experiments were performed in triplicate and average cellviability calculated as measured background corrected absorbance dividedby the absorbance of untreated control wells. EC₅

values were determined by fitting data to the four-parameter logisticequation.

The EC

values for various cytotoxic compounds and conjugates against CAIXexpressing SKRC52 cells were as follows:

-   -   Condition (a): 14 55±5 μM; 7a 21±7 μM; 7b 96±33 μM; DM1 4.3±0.5        μM; DM1SMe 0.5±0.0 μM; 9a 41±9 μM; 9b 31±6 μM.    -   Condition (b): 14 1.0±0.1 nM; 7a 0.7±0.1 nM; 7b 1.4±0.3 nM; DM1        45±10 nM; DM1SMe 5.8±1.1 nM; 9a *; 9b *.

These results show that the conjugates 7a and 9a according to theinvention release the cytotoxic drug moiety in effective amounts over 72hours under Condition (a). However, DM1 conjugates 9a and 9b did notexhibit sufficient cytotoxicity for measurement of EC

values in the 1 hour timescale of Condition (b) at concentrations up to300 nM, reflecting the relatively long half-life of these conjugates asfurther discussed below.

Ligand Binding Analysis by Flow Cytometry

Cells were detached from culture plates using a 50 mM EDTA solution inphosphate buffered saline (PBS) pH 7.4, counted and suspended to a finalconcentration of 1.5×10⁶ cells mL⁻¹ in a 1% v/v solution of FCS in PBSpH 7.4. Aliquots of 3×10⁵ cells (200 μL) were spun down and resuspendedin solutions of IRDye750 (Licor) labeled ligands (30 nM) in a 1% v/vsolution of FCS in PBS pH 7.4 (200 μL) and incubated on ice for 1 h.Cells were washed once with 200 μL 1% v/v solution of FCS in PBS pH 7.4(200 μL, spun down, resuspended in 1% v/v solution of FCS in PBS pH 7.4(300 μL) and analyzed on a FACS Canto flow cytometer (BD Bioscience).FlowJo Version 8.7 (Treestar) was used for data analysis andvisualization. Results were as follows:

(1) Flow Cytometry Analysis of Binding of Ligand-IRDye750 Conjugates1c-6c (FIG. 1 ) to CAIX Expressing SKRC52 Cells.

Cells were detached with EDTA, treated with 30 nM dye conjugate for 1 hat 0° C. washed and analyzed. Only 1c binds strongly enough to result ina strong fluorescence shift after washing of cells. Given their higher K

conjugates 2c-5c probably dissociate too quickly to be detected.Conjugate 6c, which lacks a ligand for CAIX, only shows little residualbinding.

(2) Flow Cytometry Analysis of Binding of Ligand-IRDye750 Conjugates1c-6c (FIG. 1 ) to CAIX Negative HEK Cells.

Cells were detached with EDTA, treated with 30 nM dye conjugate for 1 hat 0° C. washed and analyzed. In the absence of a specific bindinginteraction, there is little difference between cells treated with CAIXligand-dye conjugates and untreated cells.

(3) Flow Cytometry Analysis of Binding of Ligand-Alexa546 Conjugates 1band 6b to (a) CAIX Expressing SKRC52 and (b) HEK Cells Lacking CAIX ontheir Cell Surface.

Cells were detached with EDTA, treated with 30 nM dye conjugate for 1 hat 0° C. washed and analyzed. (a) Only the conjugate bearing a ligandfor CAIX can bring about an increase in fluorescence intensity. Theconjugate lacking the ligand does not give rise to a shift relative tountreated cells. (b) As expected in the absence of a cell surfacereceptor none of the conjugates can bring about a shift in fluorescenceintensity to the right.

Ligand Internalization Analysis by Flow Cytometry

SKRC52 or A549 cells were seeded in 6-well plates in their appropriateculture medium (2 mL) at a density of 1.5×10⁵ cells per well and allowedto grow for 24 h under culture conditions. The medium was replaced withmedium containing IRDye750 (Licor) labeled probes 1c or 6c (2 mL, 30 nM)and plates incubated for 1, 2 or 4 h under standard culture conditions.After washing with PBS pH 7.4 (2×2 mL). Trypsin-EDTA 0.05% (500 μL,Invitrogen) was added and plates incubated under standard cultureconditions for 15 min. Medium (500 μL) was added, cells pelleted andresuspended in PBS pH 7.4 containing 1% v/v FCS (150 μL). Afterincubating for 15 min on ice, cells were labeled for 30 min on ice withrabbit anti human CAIX IgG (1:100. Santa Cruz) in PBS pH 7.4 containing1% FCS (150 μL), washed with PBS pH 7.4 containing 1% FCS (2×150 μL) andlabeled for 30 mm on ice with goat anti rabbit IgG Alexa488 conjugate(1:100, Invitrogen) in PBS pH 7.4 containing 1% FCS (150 μL). Afterwashing with PBS pH 7.4 containing 1% FCS (2×150 μL), cells werepelleted and resuspended in PBS pH 7.4 containing 1% v/v FCS andpropidium iodide (300 μL, 1 μg mL⁻¹, Invitrogen) and analyzed on a FACSCanto flow cytometer (BD Bioscience). FlowJo Version 8.7 (Treestar) wasused for data analysis and visualization. To inhibit uptake mechanisms,cells were pre-incubated with medium containing 0.2% NaN₃ for 1 h andthe NaN₃ concentration maintained throughout the entire experiment.Alternatively, all steps were performed at 0° C. or an excess of AAZ(100 μM) was added to the culture medium.

CAIX positive SKRC52 cells attached to culture plates were incubatedwith medium containing 30 nM 1c for 1 h at 37° C. Detachment withtrypsin resulted in cells with higher fluorescence intensity than cellstreated with non-binding conjugate 6c or untreated cells. b) Aliquots ofthe same cells which had been treated with 1c and detached with trypsinwere stained with an anti CAIX antibody (aCAIX AB) followed by anAlexa488 labeled secondary antibody (2° AB) at 0° C. which upon flowcytometry analysis gave a superimposable histogram to cells treated withsecondary antibody only. We concluded that trypsin treatment had removedall surface bound CAIX and the fluorescence shift of 1c labeled cells ina) must come from internalized conjugate. Cells detached from solidsupport using EDTA and stained as before gave rise to a 10× shift influorescence intensity to the right compared to baseline giving usconfidence that CAIX detection by FCAS did indeed work.

To further support the claim that we were observing active uptakeprocesses, we decided to repeat the experiment under conditionsinhibiting uptake. SKRC52 cells were pre-treated with medium containing0.2% w/v NaN₃, for 1 h before incubation with 30 nM 1c and 0.2% w/v NaN₃at 37° C. for 1 h. NaN₃ is known to be an inhibitor of active uptakeprocesses^([1]) and indeed fluorescent signal was shifted to baseline.The same effect was achieved when incubating with 1c in the presence ofexcess AAZ as a competitive ligand or when incubating at 0° C. whichalso inhibits active uptake processes.

Extending the incubation time with 30 nM 1c at 37° C. from 1 h to 2 hand 4 h we did not see a markedly increased signal. We thus concludedthat although some internalization takes place, it is inefficient overtime.

Finally, active uptake of CAIX binding conjugate 1c into CAIX negativeA549 cells was tested as above. Since no shift in fluorescence intensityover baseline was observed, it was concluded that 1c was not taken upinto A549 cells. This is expected in the absence of a cell surfacereceptor for 1c.

Ligand Internalization Analysis by Confocal Microscopy

SKRC52 cells were seeded into 4-well cover slip chamber plates(Sarstedt) at a density of 10⁴ cells per well in RPMI medium (1 mL,Invitrogen) supplemented with 10% FCS, AA and4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 10 mM) andallowed to grow for 24 h under standard culture conditions. The mediumwas replaced with medium containing 1b or 6b (30 nM), after 1 h Hoechst33342 nuclear dye (Invitrogen) was added and randomly selected coloniesimaged on an Axiovert 200M confocal microscope (Zeiss).

Stability Determination by Mass Spectrometry

Targeted carbonate or carbamate 7a or 8a (30 μg) was dissolved in PBS pH7.4 (1.5 mL) and incubated at 37° C. under gentle agitation. Aliquots(100 μL) were removed at different time points and diluted 1:1 with aninternal standard of etodolac (TC1 Chemicals) in MeOH (20 μg mL⁻¹).Small molecules were separated from salts using an Oasis WAX onlinesample preparation column (Waters) on an Alliance HT separation module(50 μL injections, 0.3 mL min⁻¹ 0.1% aq. HCOOH for 3 min followed by 0.3mL min⁻¹ MeCN for 7 min, Waters) and analyzed by mass spectrometry/massspectrometry (MS/MS) on a Quattro API spectrometer (Waters) monitoringappropriate multiple reaction monitoring (MRM) transitions for 8a, 8band etodolac as standard. Measurements were performed in triplicate,peaks integrated and the fraction of intact test compounds calculated asfraction of signal at time t divided by signal at time zero. Sincesignals due to etodolac were constant over time a further correctionusing the internal standard as reference was omitted. For stabilitymeasures in mouse serum, compounds were dissolved in freshly thawedmouse plasma (Invitrogen), aliquots taken at different time points anddiluted with an equal volume of MeCN. After vigorous vortexting for 1min, protein precipitate was spun down and the supernatant analyzed asabove.

Half lives of 7a and 8a in mouse plasma at 37° C. as determined by massspectrometry (MS/MS) were 43 minutes and 61 minutes, respectively.Errors were <1 min.

Stability Determination by High-Performance Liquid Chromatography (HPLC)

Targeted DM1 conjugate 9a (230 μg, 140 nmol) was dissolved in PBS pH 7.4(1 mL) and incubated at 37° C. under gentle agitation. Aliquots (100 μL)were removed at different time points and diluted 1:1 with an internalstandard solution of etodolac (TCI Chemicals) in MeCN (20 μg mL⁻¹).Water (600 μL) was added and aliquots of this mixture (50 μL) analyzedover a Syngergi RP Polar column (150×4.6 mm, 4 μm, Phenomenex) on anAlliance HT separation module (1 mL min⁻¹ 5% MeCN in 0.1% aqueous TFA to100% MeCN over 20 min. Waters). Analytes were detected using a Water2996 photo array UV/VIS detector (Waters). Measurements were performedin triplicate, peaks integrated and the fraction of intact testcompounds calculated as fraction of signal at time/divided by signal attime zero. Since signals due to etodolac were constant over time afurther correction using the internal standard as reference was omitted.For stability measures in mouse serum, compounds were dissolved infreshly thawed mouse plasma (Invitrogen), aliquots taken at differenttime points and diluted with an equal volume of MeCN. After vigorousvortexting for 1 min, protein precipitate was spun down and thesupernatant analyzed as above.

As expected, the carbonate 7a (t_(1/2)=15 h) was less stable in PBS at37° C. than the carbamate 8a (t_(1/2)>24 h). No decomposition wasobserved for the DM1 conjugate 9a under the same conditions (FIG. 8 ).The stability of 7a and 8a was reduced in mouse serum in vitro(t_(1/2)=43 and 61 min respectively), but occurred in a time rangecompatible with the preferential accumulation of the AAZ conjugates atthe tumor site. The DM1 conjugate 9a was significantly more stable inmouse serum (t_(1/2)=20 h).

Animal Studies

All animal experiments were conducted in accordance with Swiss animalwelfare laws and regulations under the license number 42/2012 granted byVeterinaeramt des Kanton Zurich.

Implantation of Subcutaneous SKRC52 Tumors

SKRC52 cells were grown to 80% confluence and detached with Trypsin-EDTA0.05% (Invitrogen). Cells were washed with PBS pH 7.4 once, counted andresuspended in PBS to a final concentration of 6.7×10⁷ cells mL⁻¹.Athymic balb/c nu/nu mice, 8-10 weeks of age (Charles River) wereanesthetized with isofluorane and aliquots of 1×10⁷ cells (150 μL ofsuspension) injected subcutaneously into their lower back.

IVIS Imaging

Mice bearing subcutaneous SKRC52 tumors (200-300 mm³ in size) wereinjected intravenously with IRDye750 (Licor) labeled CAIX ligands 1c-4c(up to 10 nmol) dissolved in 5% v/v DMSO in PBS pH 7.4 (150 μL). Micewere anesthetized with isoflurane and in vivo fluorescence imagesacquired on an IVIS Spectrum imaging system (Xenogen, exposure 1s,binning factor 8, excitation at 745 nm, emission filter at 800 nm, ƒnumber 2, field of view 13.1). Images were taken after 1 h, 2 h, 4 h, 8h and 12 h and 24 h. Food and water was given ad libitum during thatperiod.

Near infrared images of SKRC52 were obtained from xenograft bearing mice1-12 h after intravenous injection of 3 nmol ligand-IRDye750 conjugates1c-5c and untargeted conjugate 6c as negative control (see FIG. 1 forstructures). Only the AAZ conjugate 1c gave good tumor to backgroundcontrast and was thus selected as a basis for further development of atargeted conjugate.

Already 1 h after the intravenous injection of 1 nmol 1c the tumor couldclearly be seen against background. The injection of 3 nmol gives astronger and longer lasting signal with good tumor to backgroundcontrast at early time points and was thus used for further imagingstudies. A dose of 10 nmol saturates the fluorescence detector with theparameters used at early time points but leads to an even longer lastingsignal.

After administration of 2c the tumor was barely visible; all otherconjugates did not reach the tumor in levels above backgroundfluorescence. Untargeted conjugate 6c also does not reach the tumor andis also cleared faster from the animal than ligand-IRDye750 conjugates.

Mice were subsequently sacrificed by cervical dislocation. Heart, lung,kidney, liver, spleen, a section of the intestine (100-150 mg), skeletalmuscle (100-150 mg) and the tumor were extracted and imaged individuallyusing above parameters. Qualitatively, a decrease in targetingperformance from 1c to 5c could be observed and very little tumor ororgan accumulation from untargeted conjugate 6c. This confirms thatbinding affinity of the targeting ligand for CAIX is an importantdeterminant for accumulation inside the tumor and in vitro profiling ofdye conjugates by FP and flow cytometry has predictive value for in vivotargeting performance.

Biodistribution Analysis

Mice (groups of 3 per time point and compound) bearing subcutaneousSKRC52 tumors (200-300 mm³ in size) were injected intravenously withIRDye750 (Licor) labeled probes 1c or 6c (3 nmol) dissolved in 5% v/vDMSO in PBS pH 7.4 (150 μL). After 1 h, 2 h or 4 h animals weresacrificed, organs extracted as above, cut into small pieces, weighedand suspended in 1:1 w/v organ homogenization buffer containing EDTA (40mM), proteinase K (6 mg/ml), Triton X-100 (1.6 μl/ml) and trace amountsof DNase 1 in PBS pH 7.4 (100 μL per 100 mg of tissue). The suspensionwas homogenized on a TissueLyser organ homogenizer (Quiagen, 25 Hz, 10min), incubated for 2 h at room temperature and 100 μL of the homogenatetransferred to a black 96-well plate. A standard dilution series of 1cin homogenization buffer (750 nM-47 nM, 25-1.5% ID g⁻¹ in steps of 1:2)was spotted alongside the organ samples in triplicate. Fluorescentimages of plates were recorded on an IVIS Spectrum imaging system(Xenogen, parameters as above) and analyzed using Living Image softwareversion 4.3.1 (Caliper Life Science) using the built-in region ofinterest (ROI) tools. Dye concentrations in organ samples in % ofinjected dose per gram of tissue (% ID g⁻¹) were inferred fromfluorescence intensities originating from the corresponding well bycomparison with the standard dilution series.

The results are shown graphically in FIGS. 3 and 4 . FIG. 3 shows organaccumulations are reported in units of percent of injected dose per gramof tissue (% ID g⁻¹), a) 1 h after intravenous administration of 3 nmol1c (blue) and 6c (red) b) 2 h after intravenous administration of 3 nmol1c (blue) and 6c (red) c) 4 h after intravenous administration of 3 nmol1c (blue) and 6c (red) d) Calibration curve (average of triplicates) forthe conversion of fluorescence intensity to % ID g⁻¹. Error barsindicate standard deviations. All data points are averages of threemice.

Accumulation of 1c in the tumor was rapid and efficient with 13.4±3.0%of injected dose per gram of tissue (% ID g⁻¹) after only 1 h (FIG. 2 b, Supporting FIGS. 10 and 11 ). This result compares favorably withprevious work on antibody-based targeting of CAIX expressing tumors,where only markedly lower tumor uptake values (a maximum of 2.4 t 0.2%ID g⁻¹) could be detected.^([27]) In our case, the dye conjugate 1c,however, progressively dissociated from the tumor (residence t_(1/2)≈1h), suggesting that an improvement of CAIX binding affinity may furthercontribute to efficient tumor targeting performance.

A tumor-to-blood ratio of 13.8:1 was observed 1 h after intravenousinjection of 1c and further improved to 79.2:1 after 4 h. Tumor-to-organratios for excretory organs ranged between 0.2:1 for liver and 14:1 forkidneys after 1 h but a high level of selectivity was observed for otherorgans (e.g., 27.6:1 for tumor to heart after 1 h). Since AAZ is a CAligand with broad isoform selectivity¹, the observed differential uptakepatterns are probably strongly influenced by relative CA expressionlevels in different tissues and the accessibility of the antigen (e.g.,intracellular CAII can be expected to be inaccessible to our chargedmolecules). Importantly, tumor targeting was clearly dependent on theCAIX-binding moiety, as revealed by the 22-fold higher tumoraccumulation at 1 h of the AAZ-based targeted dye conjugate 1c comparedto the non-targeted dye 6c. Assuming that 6c is a good model for thetissue distribution of “naked” (i.e., untargeted) anticancer agents,this comparison highlights the potential impact of ligand-based drugdelivery of therapeutically relevant doses of drugs into neoplasticmasses.

FIG. 4 shows biodistribution analysis of 1c in balb/c nu/nu mice bearingsubcutaneous SKRC52 tumors including stomach and blood values 1, 2 and 4h after giving 3 nmol of the dye conjugate intravenously. Organaccumulations are reported in units of % ID g⁻¹. Error bars indicatestandard deviations. Data shown are averages of three mice.

Analysis of Tumor Penetration

Mice bearing subcutaneous SKRC52 tumors (200-300 mm³ in size) wereinjected intravenously with Alexa546 (Invitrogen) labeled probes 1b or6b (50 nmol) dissolved in PBS pH 7.4 (150 L). After 1 h, 2 h or 4 hanimals were injected with a solution of Hoechst 33342 (Invitrogen, 5.4mM) in saline (150 μL) and sacrificed after 5 min. Organs were extractedas above and flash-frozen in Neg-50 cryo medium (Thermo Scientific)using liquid nitrogen. After warming to −20° C., samples were cut intosections of 10 μm width and directly imaged on an Axioskop 2fluorescence microscope (Zeiss).

It was found that although the conjugate has already started penetratinginto the tumor after 30 min, staining of the tumor with 1b is initiallyhighest in well-perfused areas. Later, the staining becomes morehomogeneous. After 2 h the staining becomes weaker as the conjugate isstarting to get washed out of the tumor. Fluorescence due to 6b cannotbe detected inside the tumor, which is in accordance with the lack ofmacroscopic accumulation observed with 6c.

Microscopic analysis of organs showed strong fluorescence due to 1binside the tumor and the intestine. The latter probably is due tohepatobilary excretion of the dye conjugate. An observed layer offluorescence in the stomach most likely corresponds to gastric mucosalepithelial cells, which express CAIX under normal conditions. Kidney andliver also showed some fluorescence as a result of conjugate excretionthrough these organs.

Dosage

Estimation of the recommended therapy dose of a) 7a and b) 8a in nudemice was performed by studying different dosages using a schedule offive injections on five consecutive days compared to vehicle (5% DMSO inPBS pH 7.4). The dosage regimens were 0.4 nmol/day, 1.3 nmol/day, 4.0nmol/day and 13.3 nmol/day. One mouse was used for each regimen. Whenthe animal did not lose more than 5% of its initial body weight over 15days after the initial injection, it was assumed, that the dose was welltolerated.

The study showed that 7a was tolerated up to 4.0 nmol/day, but poorlytolerated at 13.3 nmol/day. The study further showed that 8a was welltolerated up to and including 13.3 nmol/day.

Estimation of the recommended therapy dose and schedule of DM1 conjugate9a was studied in SKRC52 tumor bearing nude mice. One mouse was used fortesting each dosing scheme. Injections were given daily starting on day0 in 5% DMSO in PBS pH 7.4 (150 μL). The results are shown in FIG. 5 .Six doses of 60 nmol 9a were tolerated with only minimal weight loss.Since the animals in this study weighed on average 18% less than thoseused in the therapy study, a dose of 70 nmol per injection was used forthe therapy experiment. The number of injections was also increased from6 to 7 on 7 consecutive days.

Therapy Experiments

SKRC52 xenograft tumors were implanted into balb/c nu/nu mice (CharlesRiver) as described above. After 14 days, mice were randomly assignedinto therapy groups of 5 or 6 animals and treatment started. 5 doses of4 nmol 7a,b, 8a,b or 7 doses of 70 nmol 9a,b each in PBS pH 7.4 (150 μL)containing 5% DMSO were given on 5 or 7 consecutive days and one groupwas treated with vehicle (5% DMSO in PBS pH 7.4). In the case of 7-9b anequimolar amount of AAZ was added to the injection solution to controlfor a possible antitumor activity of CAIX inhibitors. Sorafenib andsunitnib were administered at a standard dose of 30 mg/kg as describedpreviously.^([5]) Animals were weighed and tumor sizes measured dailyand the tumor volume calculated according to the formula (longside)×(short side)²×0.5. Animals were sacrificed when the body weightfell by more than 15% relative to the first therapy day or when tumorsreached a volume of >2000 mm³. Prism 6 (GraphPad Software) was used fordata analysis (regular two-way ANOVA with the Bonferroni test).

Results are shown in FIGS. 6 and 7 . Error bars give standard errors.The therapeutic results obtained with the duocarmycin-AAZ conjugatesonly indicated a modest tumor growth inhibition effect (FIG. 6 a ).Nevertheless, targeted carbonate 7a gave rise to statisticallysignificant tumor growth retardation compared to mice that only receivedvehicle (p<0.0001) and mice receiving untargeted conjugate 7b plusequimolar amounts of AAZ (p<0.05). The carbamate-based constructs 8a and8b did not lead to any retardation in tumor growth. It seems reasonablethat the low affinity of 8a towards the antigen (K

=40.3±2.6 nM versus K

=7.3±0.5 nM for 7a) and inefficient extracellular activation may havebeen partly responsible for this effect. The treatment could beperformed with a weight loss lower than 15% of body weight (FIG. 6 b ).

For the DM1 conjugate 9a, a potent anti-tumor effect was observed atdoses, which gave only minimal toxicity (i.e., no detectable body weightloss giving 7×70 nmol of DM1-conjugate 9a on 7 consecutive days). Duringthe treatment period tumors shrank and continued to reduce in volume for7 additional days. Only 20 days after the start of treatment, tumorsstarted regrowing, as a consequence that mice had not received anyadditional drug treatment. Importantly, neither sorafenib nor sunitinib,which represent the most commonly used chemotherapeutic agents for thetreatment of kidney cancer, exhibited any detectable antitumor effect,in line with previous reports in different models of kidney cancer.These findings suggest that the targeted delivery of potent cytotoxicagents may provide a therapeutic advantage compared to the currentstandard of care. DM1 may be a particularly suitable payload for thedevelopment of targeted cytotoxics, since the presence of e.g., an estermoiety in its structure may facilitate its detoxification inclearance-related organs, thus sparing healthy tissues.

(B) Bivalent Binding Moieties

A comparative study of the tumour targeting performance of monovalentand bivalent ligands to carbonic anhydrase IX (CAIX) in renal cellcarcinoma was performed as follows.

Synthesis of Fluorescence Labeled Targeting Ligands

Monovalent acetazolamide (AAZ) derivative B1 and bivalent AAZ derivativeB2 having the structures shown in FIGS. 8 and 9 were synthesised usingstandard Fmoc solid phase peptide chemistry. These binding moieties werefluorescence labelled with IRDye 750 to provide fluorescence labelledmonovalent and bivalent ligands B3 and B4. The synthesis methods were asfollows.

Synthesis of AAZTL-B1

Commercially available polystyrene Wang p-nitrophenyl carbonate resin(250 mg, 0.15 mmol) was swollen in DMF (5 mL for 5 min) and reacted witha solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (250 μL), DIPEA(500 μL) and DMAP (2.5 mg) in DMF (4.5 mL) for 12 h at room temperatureunder shaking. The resin was washed with DMF (3×5 mL for 1 min), MeOH(3×5 mL for 1 min) and again DMF (3×5 mL for 1 mm). A solution of5-azido valeric acid (65 mg, 0.45 mmol), HATU (171 mg, 0.45 mmol) andDIPEA (148 μL, 0.9 mmol) was prepared and immediately reacted with theresin for 1 h at room temperature under shaking. After washing with DMF(6×1 min×5 mL) a solution of CuI (2.9 mg, 0.015 mmol), TBTA (8 mg, 0.015mmol) and alkyne 10 (123 mg, 0.45 mmol) in a mixture of DMF (1 mL) andTHF (1 mL) was prepared and reacted with the resin for 24 h at roomtemperature. After washing with DMF (3×1 min×5 mL), 50 mM aq. EDTAsolution (3-1 min×5 mL), DMF (3×1 min×5 mL) and DCM (3×1 min×5 mL), thecompound was cleaved by agitating the resin with a mixture of TFA (2.2mL), TIS (50 L). H₂O (50 μL), m-cresol (100 μL) and thioanisol (100 μL)for 2 h at room temperature. The resin was washed with TFA (1×5 mm×2.5mL) and the combined cleavage and washing solutions added drop-wise toice cold diethyl ether (100 mL). The precipitate was collected bycentrifugation and the product purified by reversed-phase HPLC (95% A/5%B to 20% A/80% B over 20 min). After lyophilisation the title compoundwas collected as a white powder (78 mg, 0.14 mmol, 95%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=13.01 (s, 1H), 8.32 (s, 2H), 7.89-7.82(m, 5H), 4.28 (t, J=7.0 Hz, 2H), 3.58-3.50 (m, 6H), 3.38 (t, J=6.1 Hz,2H), 3.18 (m, 2H), 3.00 (m, 2H), 2.65 (t, J=7.5 Hz, 2H), 2.59 (t, J=7.4Hz, 2H), 2.09 (t, J=7.4 Hz, 2H), 1.94 (m, 2H), 1.75 (m, 2H), 1.42 (m,2H); ¹³C-NMR (125 MHz, DMSO-d₆) δ [ppm]=172.5, 172.4, 164.8, 161.5,146.4, 122.4, 70.1, 69.8, 69.6, 67.1, 49.4, 39.1, 38.8, 35.0, 35.7,29.8, 24.8, 24.6, 22.6; HRMS: (m/z) [M+H]⁺ calcd. for C₁₉H₃₄N₉O₆S₂548.2068; found 548.2071.

Synthesis of B2

Commercially available polystyrene Wang p-nitrophenyl carbonate resin(500 mg, 0.3 mmol) was swollen in DMF (5 mL for 5 min) and reacted witha solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (500 μL), DIPEA(500 μL) and DMAP (5 mg) in DMF (4 mL) for 12 h at mom temperature undershaking. The resin was washed with DMF (3×5 mL for 1 min), MeOH (3×5 mLfor 1 min) and again DMF (3×5 mL for 1 min). A solution ofFmoc-Lys(Fmoc)-OH (532 mg, 0.9 mmol), HBTU (341 mg, 0.9 mmol), HOBt (138mg, 0.9 mmol) and DIPEA (298 μL, 1.8 mmol) was prepared and immediatelyreacted with the resin for 1 h at room temperature under shaking. Afterwashing with DMF (6×1 min×5 mL) the Fmoc group was removed with 20%piperidine in DMF (1×1 min×5 min and 2×10 min×5 mL) and the resin washedwith DMF (6×1 min×5 mL) before the next coupling step was initiated. Inthe following, the peptide was extended with Fmoc-Asp(OtBu)-OH twicefollowed by 5-azido-valerate. For this purpose, a solution of acid (1.2mmol). HATU (465 mg, 1.2 mmol) and DIPEA (397 μL, 2.4 mmol) was preparedin DMF (5 mL) and reacted with the resin for 1 h at room temperatureunder gentle agitation. Each coupling was followed by a washing stepwith DMF (6×1 min 5 mL) and Fmoc deprotection as described above. Aftercoupling of the azide, a solution of CuI (76 mg, 0.12 mmol), TBTA (21mg, 0.12 mmol) and alkyne 10 (329 mg, 1.2 mmol) in a mixture of DMF (2.5mL) and THF (2.5 mL) was prepared and reacted with the resin for 48 h atroom temperature. After washing with DMF (3×1 min×5 mL), 50 mM aq. EDTAsolution (3×1 min×5 mL), DMF (3×1 min×5 mL) and DCM (3×1 min×5 mL), thecompound was cleaved by agitating the resin with a mixture of TFA (4.4mL), TIS (100 μL), H₂O (100 μL), m-cresol (200 μL) and thioanisol (200μL) for 2 h at room temperature. The resin was washed with TFA (1×5min×5 mL) and the combined cleavage and washing solutions addeddrop-wise to ice cold diethyl ether (100 mL). The precipitate wascollected by centrifugation, dissolved in aq. MeCN and lyophilised toyield the title compound as an off-white powder (468 mg, 0.3 mmol,quant.).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=13.09 (s, 2H), 8.37 (s, 4H), 8.29-8.26(m, 3H), 8.14 (d, J=8.0 Hz, 1H), 7.91 (s, 2H), 7.80-7.78 (m, 3H), 7.71(d, J=8.0 Hz, 1H), 7.65 (t, J=5.4 Hz, 1H), 4.60-4.48 (m, overlaps withbroad H₂O peak), 4.33 (t, J=7.0 Hz, overlaps with broad H₂O peak),4.19-4.13 (m, overlaps with broad H₂O peak), 3.64-3.59 (m, 6H), 3.44 (t,J=6.3 Hz, 2H), 3.27-3.23 (m, 2H), 3.05-3.00 (m, 4H), 2.77-2.48 (m,overlaps with solvent peak), 2.20 (t, J=7.2 Hz, 4H), 2.04-1.96 (m, 4H),1.86-1.78 (m, 4H), 1.74-1.63 (br m, 1H), 1.61-1.16 (br m, 9H); HRMS:(m/z) [M+H]⁺ calcd. for C₅₄H₈₃N₂₂O₂₃S₄ 1535.4879; found 1535.4868.

Synthesis of B3

To IRDye750 NHS ester (100 μg, 84 nmol) in DMSO (10 μL) and DMF (100 μL)was added acetazolamide derivative B1 (200 μg, 366 nmol) in DMSO (20 μL)and DIPEA (2 μL, 12 μmol). The mixture was allowed to stand at roomtemperature for 2 h and then directly purified over reversed-phase HPLC(95% A/5% B to 40% A/60% B over 30 min). Fractions containing dyeconjugate were identified through their characteristic UV/VIS spectrum(λ_(max)=750 nm), pooled, lyophilised and dissolved in DMSO (50 μL) togive a dark green stock solution. Its concentration and the reactionyield were determined by measuring the absorbance at 750 nm (ε₇₅

=260,000 M⁻¹ cm⁻¹) of stock samples diluted 1:200 into PBS pH 7.4 (640μM, 32 nmol, 38%).

HRMS: (m/z) [M+4H]⁺ calcd. for C₆₈H₉₂N₁₁O₁₉S₆ 1558.4890; found1558.4844.

Synthesis of B4

To IRDye750 NHS ester (100 μg, 84 nmol) in DMSO (10 μL) and DMF (100 μL)was added B2 (200 μg, 130 nmol) in DMSO (20 μL) and DIPEA (2 μL, 12μmol). The mixture was allowed to stand at room temperature for 2 h andthen directly purified over reversed-phase HPLC (95% A/5% B to 40% A/60%B over 30 min). Fractions containing dye conjugate were identifiedthrough their characteristic UV/VIS spectrum (=750 nm), pooled,lyophilised and dissolved in DMSO (50 μL) to give a dark green stocksolution. Its concentration and the reaction yield were determined bymeasuring the absorbance at 750 nm (ε₇₅₀₀=260,000 M⁻¹ cm⁻¹) of stocksamples diluted 1:200 into PBS pH 7.4 (287 μM, 14 nmol, 17%). HRMS:(m/z) [M+4H]⁺ calcd. for C₁₀₃H₁₄₁N₂₄O₃₀₆S % 2545.7700; found 2545.7703.

Binding Performance by Surface Plasmon Resonance

Binding experiments of monovalent and bivalent AAZ derivatives to CAIXusing surface plasmon resonance indicated a fast association for bothcompounds (k₀=1.48-10⁶ M⁻¹s⁻¹ and k₀₁=1.28×10⁶ M⁻¹s⁻¹, k₀₂=1.36×10⁶ RU⁻¹respectively). Whilst monovalent ligand B1 completely dissociated fromthe CAIX-coated surface within seconds (k_(d)=0.015 s⁻¹, K_(d)=10.5 nM),bivalent compound B2 exhibited no apparent dissociation and could onlybe removed with harsh acid treatment (FIG. 1 b ).

Binding Performance by Flow Cytometry

Flow cytometry was performed as described above with monovalent andbivalent near infrared dye conjugates B3 and B4 and negative controlconjugates lacking the ligand on CAIX-positive SKRC52 cells andCAIX-negative HEK cells⁵. The results indicated a clear ligand- andreceptor-dependent binding to cells. The shift in fluorescence intensityfor bivalent conjugate B4 was more pronounced than the one observed formonovalent B3, which is consistent with the results obtained from SPR.

In Vivo Investigation of Targeting Performance

Further studies tested the ability of near infrared dye conjugates B3and B4 to localise to CAIX-expressing SKRC52 xenografts in vo. Both dyeconjugates strongly accumulated in the tumour, as revealed by wholeanimal near infrared fluorescence imaging and by analysis of theextracted organs. While the initial clearance profile was comparable forboth targeted molecules, the bivalent conjugate B4 exhibited asignificantly longer residence on the tumour. Twenty-four hours afterinjection, the integrated fluorescence signal in the tumour frombivalent conjugate B4 was 40%, while the monovalent conjugate B3 haddecayed to 14% of its initial value (p=0.002; unpaired two-sided t-test;Supplementary FIG. 4 ).

To gain a better understanding of the absolute tumour uptake ofmonovalent dye conjugate B3 compared to bivalent B4 and tumour to organselectivity, organs were extracted, tissues homogenised and fluorescenceintensity measured on a per gram basis. Comparison to a standarddilution series of IRDye750 in organ homogenate allowed the measurementof absolute uptake levels into organs, as percent injected dose per gram(% ID g⁻¹). Bivalent dye conjugate B4 exhibited a >3-fold higherabsolute accumulation in tumours compared to monovalent B3 at 24 h(5.3±0.6 versus 1.4±0.6% IDg⁻¹). Compound B4 thus compares veryfavourably with recently described monoclonal antibodies against CAIX.While uptake into heart, spleen, muscle and circulation in bloodrelative to tumour was low (tumour:organ >30), slightly lower tumour toorgan ratios were observed for kidneys and stomach for both conjugates.Interestingly, tumour:liver and tumour:intestine ratios were lower formonovalent B3 than for bivalent B4 whilst B4 exhibited a highertumour:lung ratio than B3.

Synthesis of Drug Conjugates

Targeted and untargeted drug conjugates B7 and B8 having the structuresshown in FIG. 10 were prepared as follows. Compound B7 is a bivalentconjugate according to the present invention and has the same bivalenttargeting scaffold as B2 and B4. B8 is a reference example having asimilar scaffold but no AAZ targeting ligands.

Synthesis of B7

Bivalent targeted linker B11 (20 mg, 13 μmol), TCEP·HCl (7.6 mg, 27μmol) and DIPEA (2 μL) were dissolved in degassed DMF (500 μL). After 1h 2,2′-dipyridyldisulphide (11.7 mg, 53 μmol) was added. The mixture wasstirred at room temperature for 12 h, diluted with NMP (500 μL) and wasadded drop wise to ice cold diethyl ether (40 mL). The precipitate wascollected by centrifugation, re-dissolved in DMF (200 μL) and NMP (200μL) and precipitated again with ice cold diethyl ether (40 mL) and driedunder vacuum to give the activated disulphide as a white residue (18 mg,11 μmol, 85%). An aliquot of the activated disulphide (15 mg, 9 μmol)was dissolved in DMF (400 μL) and DM1 free thiol (7 mg, 9 μmol) added.The reaction was allowed to stand at room temperature for 48 h afterwhich the product was recovered by reversed phase HPLC (95% A/5% B to20% A/80% B over 20 min). Fractions containing the desired product by MSwere pooled and lyophilised to yield the title compound as an off whitepowder (9.5 mg, 4 μmol, 47%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=12.98 (s, 2H), 8.31 (s, 4H), 8.22-8.15(m, 4H), 8.07 (d, J=8.2 Hz, 1H), 7.85 (s, 2H), 7.69-7.59 (m, 2H), 7.12(s, 1H), 6.89 (s, 1H) 6.61-6.52 (m, 3H), 5.92 (br s, 1H), 5.57-5.52 (m,1H), 5.30-5.29 (m, 1H), 4.52-4.43 (m, 5H), 4.39-4.34 (m, 1H), 4.27 (t,J=6.9 Hz, 4H), 4.19-4.16 (m, 1H), 4.08-4.03 (m, 1H), 3.92-3.90 (m, 3H),3.53-2.41 (m, overlap with solvent peak), 2.13-2.12 (m, 4H), 2.04-2.01(m, 1H), 1.97-1.91 (m, 4H), 1.79-1.73 (m, 4H), 1.67-1.54 (m, 4H),1.51-1.10 (m, 21H), 0.77 (s, 3H);

HRMS: (m/z) [M+2H]²⁺ calcd. for C₁₆H₁₁₉ClN₂₄O₃₃S₆ 1122.3270; found1122.3279.

Synthesis of B8

Bivalent untargeted linker B12 (20 mg, 17 μmol), TCEP·HCl (19 mg, 68μmol) and DIPEA (10 μL) were dissolved in degassed DMF (1 mL). After 1 h2,2′-dipyridyldisulphide (22 mg, 100 μmol) was added. The mixture wasstirred at room temperature for 12 h, diluted with NMP (500 μL) and wasadded drop wise to ice cold diethyl ether (40 mL). The precipitate wascollected by centrifugation, re-dissolved in DMF (200 μL) and NMP (200μL) and precipitated again with ice cold diethyl ether (40 mL) and driedunder vacuum to give the activated disulphide as a white residue (45 mg,product+side products). An aliquot of the residue (15 mg) was dissolvedin DMF (400 μL) and DM1 free thiol (7 mg, 9 μmol) was added. Thereaction was allowed to stand at room temperature for 48 h after whichthe product was recovered by reversed phase HPLC (95% A/5% B to 20%A/80% B over 20 min). Fractions containing the desired product by MSwere pooled and lyophilised to yield the title compound as an off whitepowder (7.4 mg, 3.9 μmol, 42%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=8.22-8.09 (m, 5H), 7.83 (s, 2H),7.64-7.58 (m, 2H), 7.12 (s, 1H), 6.89 (s, 1H), 6.61-6.52 (m, 3H), 5.93(s, 1H), 5.55 (dd, J=9.1, 14.8 Hz, 1H), 5.32-5.28 (m, 1H), 4.56-4.43 (m,6H), 4.27 (t, J=6.85 Hz, 4H), 4.20-4.17 (m, 1H), 4.05 (t, J=12.2 Hz,11H), 3.91 (s, 3H), 3.49-2.41 (m, overlap with solvent peak), 2.25 (t,J=7.4 Hz, 4H), 2.15 (m, 4H), 2.04-2.02 (br m, 1H), 1.80-1.73 (m, 8H),1.62-1.10 m, 24H), 0.77 (s, 3H); HRMS: (m/z) [M+H]⁺ calcd. forC₈₂H₁₁₆ClN₁₆O₃₁S₂ 1919.7117; found 1919.7098.

Synthesis of B9

Commercially available polystyrene Wang p-nitrophenyl carbonate resin(250 mg, 0.15 mmol) was swollen in DMF (5 mL for 5 min) and reacted witha solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (250 μL), DIPEA(500 μL) and DMAP (2.5 mg) in DMF (4.5 mL) for 12 h at room temperatureunder shaking. The resin was washed with DMF (3×5 mL for 1 min), MeOH(3×5 mL for 1 min) and again DMF (3×5 mL for 1 min). A solution of5-azido valeric acid (65 mg, 0.45 mmol), HATU (171 mg, 0.45 mmol) andDIPEA (148 μL, 0.9 mmol) was prepared and immediately reacted with theresin for 1 h at room temperature under shaking. After washing with DMF(6×1 min×5 mL) a solution of CuI (2.9 mg, 0.015 mmol). TBTA (8 mg, 0.015mmol) and 5-hexynoic acid (51 mg, 50 μL, 0.45 mmol) in a mixture of DMF(1 mL) and THF (1 mL) was prepared and reacted with the resin for 24 hat room temperature. After washing with DMF (3×1 min×5 mL), 50 mM aq.EDTA solution (3×1 min×5 mL), DMF (3×1 minx 5 mL) and DCM (3×1 mm×5 mL),the compound was cleaved by agitating the resin with a mixture of TFA(2.2 mL), TIS (50 μL), H₂O (50 μL), m-cresol (100 μL) and thioanisol(100 μL) for 2 h at room temperature. The resin was washed with TFA (1×5min×2.5 mL) and the combined cleavage and washing solutions addeddrop-wise to ice cold diethyl ether (100 mL). The precipitate wascollected by centrifugation and the product purified by reversed-phaseHPLC (95% A/5% B to 20% A/80% B over 20 min). After lyophilisation thetitle compound was collected as a white powder (21 mg, 54 μmol, 36%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=7.90-7.86 (m, 5H), 4.29 (t, J=7.0 Hz,2H), 3.60-3.51 (m, 6H), 3.40 (t, J=6.1 Hz, 2H), 3.20 (q, J=5.8 Hz, 2H),3.00-2.96 (m, 2H), 2.62 (t, J=7.6 Hz, 2H), 2.26 (t, J=7.4 Hz, 2H), 2.10(t, J=7.4 Hz, 2H), 1.85-1.74 (m, 4H), 1.46-1.42 (m, 2H); ¹³C-NMR (125MHz, DMSO-d₆) δ [ppm]=174.8, 172.4, 146.7, 122.3, 70.1, 69.8, 69.5,67.2, 49.4, 39.0, 38.9, 35.0, 33.6, 29.8, 24.9, 24.8, 22.7; HRMS: (m/z)[M+H]⁺ calcd. for C₁₇H₃₂N₅O₅ 386.2398; found 386.2403.

Synthesis of B10

Commercially available polystyrene Wang p-nitrophenyl carbonate resin(500 mg, 0.3 mmol) was swollen in DMF (5 mL for 5 min) and reacted witha solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (500 μL), DIPEA(50 0 μL) and DMAP (5 mg) in DMF (4 mL) for 12 h at room temperatureunder shaking. The resin was washed with DMF (3×5 mL for 1 min), MeOH(3×5 mL for 1 min) and again DMF (3×5 mL for 1 min). A solution ofFmoc-Lys(Fmoc)-OH (532 mg, 0.9 mmol), HBTU (341 mg, 0.9 mmol), HOBt (138mg, 0.9 mmol) and DIPEA (298 μL, 1.8 mmol) was prepared and immediatelyreacted with the resin for 1 h at room temperature under shaking. Afterwashing with DMF (6×1 mm×5 mL) the Fmoc group was removed with 20%piperidine in DMF (1×1 mm×5 min and 2×10 min 5 mL) and the resin washedwith DMF (6×1 min×5 mL) before the next coupling step was initiated. Inthe following, the peptide was extended with Fmoc-Asp(OtBu)-OH twicefollowed by 5-azido-valerate. For this purpose, a solution of acid (1.2mmol), HATU (465 mg, 1.2 mmol) and DIPEA (397 μL, 2.4 mmol) was preparedin DMF (5 mL) and reacted with the resin for 1 h at room temperatureunder gentle agitation. Each coupling was followed by a washing stepwith DMF (6×1 min×5 mL) and Fmoc deprotection as described above. Aftercoupling of the azide, a solution of CuI (76 mg, 0.12 mmol), TBTA (21mg, 0.12 mmol) and 5-hexyonic acid (440 μL, 1.2 mmol) in a mixture ofDMF (2.5 mL) and THF (2.5 mL) was prepared and reacted with the resinfor 48 h at room temperature. After washing with DMF (3×1 min×5 mL), 50mM aq. EDTA solution (3×1 min×5 mL), DMF (3×1 min×5 mL) and DCM (3×1min×5 mL), the compound was cleaved by agitating the resin with amixture of TFA (4.4 mL), TIS (100 μL), H₂O (100 μL), m-cresol (200 μL)and thioanisol (200 μL) for 2 h at room temperature. The resin waswashed with TFA (1×5 min-5 mL) and the combined cleavage and washingsolutions added drop-wise to ice cold diethyl ether (100 mL). Theprecipitate was collected by centrifugation and the product purified byreversed-phase HPLC (95% A/5% B to 20% A/80% B over 20 min). Afterlyophilisation the title compound was collected as a white powder (64mg, 53 μmol 17%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=8.25-8.22 (m, 3H), 8.09 (d, J=8.1 Hz,1H), 7.85 (s, 2H), 7.78-7.73 (br m, 3H), 7.66 (d, J=7.9 Hz, 1H), 7.59(t, J=5.3 Hz, 1H), 4.55-4.44 (m, 4H), 4.29 (t, J=7.0 Hz, 4H), 4.14-4.09(m, 2H), 3.60-3.55 (m, 6H), 3.40 (t, J=6.2 Hz, 2H), 3.22-3.19 (m, 2H),3.01-2.92 (m, 4H), 2.73-2.44 (m, overlap with solvent peak), 2.26 (t,J=7.4 Hz, 4H), 2.15 (t, J=7.2 Hz, 4H), 1.85-1.74 (m, 7H), 1.70-1.60 (brin, 1H), 1.55-1.14 (br m, 9H); HRMS: (m/z) [M+H]⁺ calcd. forC₅₀₀H₇₉N₁₄O₂₁ 1211.5539; found 1211.5515.

Synthesis of B11

Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500mg, 0.415 mmol, RAPP Polymere) was swollen in DMF (3×5 min×5 mL), theFmoc group removed with 20% piperidine in DMF (1×1 min×5 mL and 2×10min×5 mL) and the resin washed with DMF (6×1 min×5 mL). A solution ofFmoc-Lys(Fmoc)-OH (736 mg, 1.25 mmol). HBTU (472 mg, 1.25 mmol), HOBt(191 mg, 1.25 mmol) and DIPEA (412 μL, 2.5 mmol) was prepared andimmediately reacted with the resin for 1 h at room temperature undershaking. After washing with DMF (6×1 mm×5 mL) the Fmoc group was removedwith 20% piperidine in DMF (1×1 min×5 min and 2×10 min×5 mL) and theresin washed with DMF (6×1 min×5 mL) before the next coupling step wasinitiated. In the following, the peptide was extended withFmoc-Asp(OtBu)-OH twice followed by 5-azido-valerate. For this purpose,a solution of acid (1.7 mmol), HATU (643 mg, 1.7 mmol) and DIPEA (549μL, 3.3 mmol) was prepared in DMF (5 mL) and reacted with the resin for1 h at room temperature under gentle agitation. Each coupling wasfollowed by a washing step with DMF (6×1 min×5 mL) and Fmoc deprotectionas described above. After coupling of the azide, a solution of CuI (106mg, 0.17 mmol), TBTA (29 mg, 0.17 mmol) and alkyne 10 (455 mg, 1.7 mmol)in a mixture of DMF (2.5 mL) and THF (2.5 mL) was prepared and reactedwith the resin for 48 h at room temperature. After washing with DMF (3×1min×5 mL), 50 mM aq. EDTA solution (3×1 min 5 mL), DMF (3×1 min×5 mL)and DCM (3×1 mm×5 mL), the compound was cleaved by agitating the resinwith a mixture of TFA (4.4 mL), TIS (100 μL), H₂O (100 μL), m-cresol(200 μL) and thioanisol (200 μL) for 2 h at room temperature. The resinwas washed with TFA (1×5 min×5 mL) and the combined cleavage and washingsolutions added drop-wise to ice cold diethyl ether (100 mL). Theprecipitate was collected by centrifugation and the product purified byreversed-phase HPLC (95% A/5% B to 20% A/80% B over 20 min). Afterlyophilisation the title compound was collected as a white powder (68mg, 45 μmol, 10%).

¹H-NMR (400 MHz, DMSO-d₆) δ [ppm]=13.01 (s, 2H), 8.32 (s, 4H), 8.21 (t,J=7.5 Hz, 3H), 8.09 (d, J=8.1 Hz, 1H), 8.05 (d, J=7.9 Hz, 1H), 7.87 (s,2H), 7.74 (d, J=7.84 Hz, 1H), 7.61 (t, J=5.4 Hz, 1H), 4.55-4.45 (m,overlap with broad water peak), 4.40-4.34 (m, overlap with broad waterpeak), 4.29 (t, J=7.0 Hz, overlap with broad water peak), 4.24-4.22 (m,overlap with broad water peak), 3.07-2.94 (br m, 2H), 2.90-2.41 (m,overlap with solvent peak), 2.15 (t, J=7.1 Hz, 4H), 1.99-1.92 (m, 4H),1.82-1.74 (m, 4H), 1.71-1.24 (br m, 10H); HRMS: (m/z) [M+H]⁺ calcd. forC₅₁H₇₄N₂₁O₂₃S₅ 1508.3864; found 1508.3861.

Synthesis of B12

Commercially available pre-loaded Fmoc-Cys(Trt) on Tentagel resin (500mg, 0.415 mmol, RAPP Polymere) was swollen in DMF (3×5 min×5 mL), theFmoc group removed with 20% piperidine in DMF (1×1 min×5 mL and 2×10min×5 mL) and the resin washed with DMF (6×1 min 5 mL). A solution ofFmoc-Lys(Fmoc)-OH (736 mg, 1.25 mmol), HBTU (472 mg, 1.25 mmol), HOBt(191 mg, 1.25 mmol) and DIPEA (412 μL, 2.5 mmol) was prepared andimmediately reacted with the resin for 1 h at room temperature undershaking. After washing with DMF (6×1 min×5 mL) the Fmoc group wasremoved with 20% piperidine in DMF (1×1 min×5 min and 2×10 min×5 mL) andthe resin washed with DMF (6×1 min×5 mL) before the next coupling stepwas initiated. In the following, the peptide was extended withFmoc-Asp(OtBu)-OH twice followed by 5-azido-valerate.

For this purpose, a solution of acid (1.7 mmol), HATU (643 mg, 1.7 mmol)and DIPEA (549 μL, 3.3 mmol) was prepared in DMF (5 mL) and reacted withthe resin for 1 h at room temperature under gentle agitation. Eachcoupling was followed by a washing step with DMF (6×1 min×5 mL) and Fmocdeprotection as described above. After coupling of the azide, a solutionof CuI (106 mg, 0.17 mmol), TBTA (29 mg, 0.17 mmol and 5-hexyonic acid(609 μL, 1.7 mmol) in a mixture of DMF (2.5 mL) and THF (2.5 mL) wasprepared and reacted with the resin for 48 h at room temperature. Afterwashing with DMF (3×1 min×5 mL), 50 mM aq. EDTA solution (3×1 min×5 mL),DMF (3×1 min×5 mL) and DCM (3×1 min×5 mL), the compound was cleaved byagitating the resin with a mixture of TFA (4.4 mL), TIS (100 μL), H₂O(100 μL), m-cresol (200 μL) and thioanisol (200 μL) for 2 h at roomtemperature. The resin was washed with TFA (1/5 min×5 mL) and thecombined cleavage and washing solutions added drop-wise to ice colddiethyl ether (100 mL). The precipitate was collected by centrifugationand the product purified by reversed-phase HPLC (95% A/5% B to 20% A/80%B over 20 min). After lyophilisation the title compound was collected asa white powder (147 mg, 0.12 mmol, 30%).

¹H-NMR (500 MHz, DMSO-d₆) δ [ppm]=8.22-8.19 (m, 3H), 8.08 (d, J=8.9 Hz,1H), 8.02 (d, J=7.8 Hz, 1H), 7.83 (s, 2H), 7.72 (d, J=7.8 Hz, 1H),7.59-7.56 (m, 1H), 4.56-4.43 (m, 3H), 4.37-4.34 (m, 1H), 4.27-4.20 (m,4H), 3.03-2.92 (m, 2H), 2.87-2.39 (m, overlap with solvent peak), 2.25(t, J=7.35 Hz, 4H), 2.13 (t, J=7.0 Hz, 4H), 1.83-1.21 (br m, 16H); HRMS:(m/z) [M+H]⁺ calcd. for C₄₇H₇₀₀N₁₃O₂₁S 1184.4524; found 1184.4508.

Properties of Drug Conjugates

Both the targeted drug B7 and untargeted B8 were equally toxic in vitro.If conjugates were internalised in a receptor-dependent fashion andactivated intracellularly, targeted conjugate B7 would be expected toaccumulate in CAIX-expressing cells and to be more toxic than untargeteddrug B8. This does not seem to be the case. The present inventors thushypothesised that the conjugate would accumulate at the tumour site,where reducing agents (e.g., glutathione released from dying cells)would cleave the disulfide bond in extracellular space and lead to drugrelease. DM1 would then diffuse into adjacent cells to act on itsintracellular target.

A preliminary dose finding study with conjugate B7 was conducted. Even adose as low as 6 nmol on 8 consecutive days led to substantial tumourshrinkage. Five doses of 48 nmol within six days completely eradicatedthe tumour but showed some toxicity. Finally, a therapeutic schedule of35 nmol on 8 consecutive days was used, which was well-tolerated inSKRC52 tumour bearing mice (FIG. 11 ). On the 12^(th) day after thestart of treatment, two mice were tumour free and the average tumourvolume for all mice had dropped from 200 mm³ initial tumour volume tobelow 50 mm³. The two mice with complete regression and one from thedose escalation study were tumour free 90 days after start of therapyand were thus considered cured. The remaining tumours regrew.Importantly, control conjugates lacking the targeting ligand, orbivalent scaffold B2 without the payload did not have a statisticallysignificant antitumor effect.

(C) Binding Moieties by Screening of a DNA-Encoded Library

Chemical technologies for the discovery of high-affinity protein bindersprovide techniques to go beyond naturally-occurring ligands for diseasetargeting applications. Combinatorial chemical libraries ofunprecedented size can be constructed and screened by tagging organicmolecules with DNA fragments, serving as amplifiable identificationbarcodes [group Liu; group Neri]. DNA-encoded chemical libraries, firstpostulated by Lerner and Brenner [REF], can be synthesized with one ortwo sets of molecules displayed at the extremities of complementary DNAstrands, yielding single- or dual-pharmacophore chemical libraries,respectively.

The present inventors have studied a novel DNA-encoded self-assemblingchemical (ESAC) library, containing 111,100 small molecules in order toidentify a new bivalent binding moiety for CAIX.

Synthesis of DNA-Encoded Self-Assembling Chemical (ESAC) Library

A dual pharmacophore ESAC library of 11,100 compounds was synthesisedusing a novel chemical strategy which allows the sequence-basedidentification and quantification of library members. The EncodedSelf-Assembling Chemical (ESAC) library was constructed by hybridizingtwo individually synthesized and purified single-stranded sublibraries Aand B. Chemical compounds carrying a carboxylic acid, anhydride,N-hydroxysuccinimide ester or isothiocyanate groups were coupled to theprimary amino group at the 5′-end (sublibrary A) or 3′-end (sublibraryB) of modified oligonucleotides to produce the library as shown in FIG.13 .

Sub-Library a Synthesis.

The synthesis of the DNA-encoded sub-library A of 550 compounds has beendescribed by Dumelin, C. E., Scheuermann, J., Melkko, S. & Neri. D. inBioconjugate chemistry 17, 366-370 (2006). In short, 48-meroligonucleotides (IBA GmbH) carrying a free amino group at the 5′-end(ω-aminohexyl phosphate diester) were reacted with activated carboxylicacid-, sulfonyl chloride- or isothiocyanate-containing building blocksto give the corresponding amide, sulfonamide and thiourea conjugates.Oligonucleotide sequences followed the pattern 5′-GGA GCT TCT GAA TTCTGT GTG CTG XXX XXX CGA GTC CCA TGG CGC AGC-3′, (SEQ ID NO: 2) where XXXXXX represents the coding sequence (6 nucleotides) that unambiguouslyidentifies each individual library member.

Sub-Library B Synthesis.

Sub-library B was built using 41-mer 3′-amino-modified oligonucleotides,which were coupled with activated Fmoc-protected amino acid, carboxylicacid, carboxylic acid anhydride and sulfonyl chloride building blocks togive the corresponding amide or sulfonamide conjugates. All librarycompounds were coupled initially to the same oligonucleotide of thesequence 5′-CAT GGG ACT CG ddd ddd CAG CAC ACA GAA TTC AGA AGC TCC-3′(SEQ ID NO: 3) (IBA GmbH), which was designed to be complementary to thesub-library A oligonucleotides and contained a 6 nucleotide abasicspacer region (d, deoxyabasic), which allows promiscuous duplexformation with the coding region of sub-library

Conjugation of Fmoc—Protected Amino Acids and Carboxylic Acids with3′-Amino-Modified Sub-Library B Oligonucleotide:

Fmoc-protected amino acids or carboxylic acids in dimethyl sulfoxide(DMSO, 12.5 μl, 100 mM), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) in DMSO (12 μl, 100 mM), N-hydroxysulfosuccinimide (S—NHS) in 2:1DMSO/H₂O, (10 μl, 333 mM) were added to DMSO (215 μl) and allowed tostand at 30° C. for 30 min. Subsequently, a mixture of amino modifiedsub-library B oligonucleotide in H₂O (5 μl, 5 nmol) and triethylaminehydrochlroide in H2O (TEA·HCl 50 μl 500 mM, pH 10.0) was added and thereaction kept at 30° C. for 12 h. Carboxylic acid conjugation reactionswere quenched with tris(hydroxylmethyl)aminomethane hydrochloride in H2O(Tris·HCl, 20 μl, 500 mM, pH 8.1) at 30° C. for 1 h. Fmoc-protectedamino acid conjugation reactions were quenched and deprotected with Trisin H2O (5 μl, 1 M) and TEA (5 μl) at 30° C. for 1 h. After quenching anddeprotection, the DNA-compound conjugate was precipitated with EtOHbefore purifying by HPLC. The separated and collectedoligonucleotide-compound conjugates were vacuum-dried overnight,redissolved in H2O (100 μl), and analysed by ESI-LC-MS31.

Conjugation of Sulfonyl Chlorides with 3′-Amino-Modified Sub-Library BOligonucleotide:

Sulfonyl chlorides in acetonitrile (MeCN, 25 μl, 100 mM) were mixed withsodium hydrogen carbonate in H2O (25 μl, 1 M, pH 9.0), MeCN (100 μl),H₂O (95 μl) and subsequently reacted with amino-modified sub-library Boligonucleotide in H₂O (5 μl, 5 nmol) at 30° C. for 12 h. The reactionwas quenched with Tris·HCl (20 μl, 500 mM, pH 8.1) at 30° C. for 1 h.After quenching the DNA-compound conjugate was precipitated with EtOHbefore purifying by HPLC. The separated and collectedoligonucleotide-compound conjugates were vacuum-dried overnight,redissolved in H₂O (100 μl), and analysed by ESI-LC-MS. The separatedand collected oligonucleotide-compound conjugates were vacuum-driedovernight, redissolved in H2O (100 μl), and analysed by ESI-LCMS

Conjugation of Carboxylic Acid Anhydrides with 3′-Amino-ModifiedSub-Library B Oligonucleotide:

Carboxylic acid anhydrides in DMSO (25 μl, 100 mM) were mixed withsodium hydrogen phosphate in H₂O (25 μl, 500 mM, pH 7.1), DMSO (195 μl),H₂O (35 μl) and subsequently reacted with amino-modified sub-library Boligonucleotide in H2O (5 μl, 5 nmol) overnight at 30° C. The reactionwas quenched with Tris·HCl (20 μl, 500 mM, pH 8.1) at 30° C. for 1 h.After quenching the DNA-compound conjugate was precipitated with EtOHbefore purifying by HPLC31. The separated and collectedoligonucleotide-compound conjugates were vacuum-dried overnight,redissolved in H2O (100 μl), and analysed by ESI-LC-MS. To unambiguouslylabel library members in sub-library B, individualoligonucleotide-compound conjugates were extended with a uniqueidentifier sequence. For this purpose, 202 39-mer code oligonucleotidesof sequence 5′-CCT GCA TCG AAT GGA TCC GTG XXX XXX XX GCA GCT GCG C-3′(SEQ ID NO:4) (IBA GmbH) were used, where XXX XXX XX denotes an 8-digitcode region. The 202 HPLC-purified oligonucleotide-compound conjugateswere ligated to these coding oligonucleotides with the help of achimeric

(DNA/RNA) adapter oligonucleotide (5′-CGA GTC CCA TGG CGC AGC TGC-3′,(SEQ ID NO: 5) bold: RNA portions), which is complementary to both, thesub-library B oligonucleotide-compound conjugates and the sub-library Bcode oligonucleotides. The adapter oligonucleotide was eventuallyremoved by RNase H (New England Biolabs) treatment.

Ligation protocol: Sub-library B oligonucleotide-compound conjugate inH₂O (50 μl, 2 μM), sub-library B code oligonucleotide in H₂O (10 μl, 15μM), sub-library B chimeric RNA/DNA adapter oligonucleotide in H₂O (10μl, 30 μM), 10×ligation reaction buffer (10 μl, New England Biolabs) andH₂O (19.5 μl) were mixed and heated up to 90° C. for 2 min before themixture was allowed to cool down to 22° C. T4 DNA ligase (0.5 μl, NewEngland Biolabs) was added and ligation performed at 16° C. for 10 hoursbefore inactivating the ligase at 70° C. for 15 min.

Library Hybridization and Code Transfer to Sub-Library a Strand.

To obtain the final library, sub-libraries A and B were firsthybridized, resulting in a combinatorial collection of duplexes, whereeach member of sub-library A could pair with each member of sub-libraryB. For the unambiguous identification of any dual pharmacophorecombination by high-throughput sequencing, coding information for A andB need to be given on the same DNA strand. This was achieved by a Klenowpolymerase assisted sublibrary A strand extension of the A/Bheteroduplexes, which transferred the coding information from thesublibrary B strand onto the sublibrary A strand Hybridization andKlenow-encoding protocol: Sub-library A in H₂O (115 μl, equimolarmixture of all library members, total concentration 864 nM), andsub-library B in H2O (100 μl, equimolar mixture of all library members,total concentration 1 μM), 10×NEB2 reaction buffer (100 μl, New EnglandBiolabs) and H₂O (685 μl) were mixed and heated up to 90° C. for 2 min,then

cooled down to 22° C. The hybridized library was purified withnucleotide removal columns (Qiagen, elution with 6×140 μl Qiagen EBbuffer on six separate columns). For Klenow-encoding, hybridized andpurified ESAC library in EB buffer (800 μl), 10×NEB2 reaction buffer(100 μl. New England Biolabs), deoxynucleotide (dNTP) solution mix (100μl, 500 μM, final concentration 50 μM, New England Biolabs) and Klenowfragment (10 μl, New England Biolabs) were mixed and incubated at 37° C.for 30 min.

Cloning, Expression and Biotinylation of CAIX.

Recombinant His6-tagged human CAIX was cloned and expressed as describedby J. K. Ahlskog et al. in British Journal of Cancer 101, 645-657(2009). The protein was chemically biotinylated with EZ-Link NHS-Biotin(Thermo Scientific) for affinity screening according to supplier'sinstructions.

Affinity Screening of the ESAC Library Against CAIX.

Affinity selections were performed using a KingFisher magnetic particleprocessor (Thermo Scientific). Streptavidin-coated magnetic beads (0.1mg) were resuspended in PBS (100 μl, 50 mM NaPi, 100 mM NaCl, pH 7.4)and subsequently incubated with biotinylated CAIX (100 μl, 0.1 μM or 1.0μM concentration) for 30 min with continuous gentle mixing CAIX-coatedbeads were washed three times with PBST (200 μl, 50 mM NaPi, 100 mMNaCl, 0.05% v/v Tween 20, pH 7.4) that was supplemented with biotin (100μM) in order to block remaining binding sites on streptavidin, andsubsequently incubated with the ESAC library (100 μl, 100 nM totalconcentration, in PBST) for 1 h with continuous gentle mixing. Afterremoving unbound library members by washing five times with PBST (200μl), beads carrying bound library members were resuspended in buffer EB(100 μl, QIAquick PCR purification kit, Qiagen) and the DNA-compoundconjugates separated from the beads by heat denaturation of streptavidinand CAIX (95° C. for 5 min). The DNA of eluted library members wasamplified by PCR, introducing at the same time additional,selection-specific DNA barcodes, and submitted to Illumina

high-throughput DNA sequencing.

In multiple selection experiments, the A-493/B-202 pair ofpharmacophores was found to be highly enriched (FIG. 14 ), compared tothe unselected library and to the other library members after CAIXselection (>200-fold enrichment):

In Vitro Binding Studies

A-493 and B-202 were first conjugated with fluorescently labelled 8-mercomplementary amino-modified locked nucleic acids (LNA™), allowed toform a heteroduplex structure and submitted to fluorescence polarizationaffinity measurements against CAIX. Fluorescence polarization (FP)measurements were performed by incubating 5 nM fluorescently labelledprobe and recombinant human Carbonic Anhydrase IX with increasingconcentrations for 1 h at 22° C. The FP was measured on a Spectra MaxParadigm multimode plate reader (Molecular Devices). On LNA™, theA-493/B-202 combination revealed a dissociation constant of 14.6 t 0.7nM, whereas B-202 (acetazolamide) alone had a K_(d) of 34.9±0.9 nM.

Synthesis of CAIX Ligands with and without Fluorophores

Next, linked chemical compounds having binding moieties with and withoutfluorophores having the structures shown in FIG. 16 were synthesizedusing standard solid-phase coupling procedures using various spacers,containing a modification site for an optional fluorophore conjugation.Representative syntheses (compounds C5a and C5c) were performed asfollows.

N-[4,4-bis(4-hydroxyphenyl)pentanoyl]-β-aspartyl-β-aspartyl-N-{2-[2-(2-aminoethoxy)ethoxy]ethyl}-6-(4-{4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino]butyl}-1H-1,2,3-triazol-1-yl-L-norleucinamide(C5a)

Commercially available pre-loaded O-bis-(aminoethyl)ethylene glycol ontrityl resin (200 mg, 0.12 mmol) was swollen first in DCM (3×5 min×2 ml)and then in DMF (3×5 min×2 ml). Fmoc-protected azidolysine (142 mg, 0.36mmol), HBTU (137 mg, 0.36 mmol). HOBt·H₂O (55 mg, 0.36 mmol) and DIPEA(119 μl, 0.72 mmol) were dissolved in DMF (2 ml), the mixture wasallowed to stand at 22° C. for 15 min and then reacted with the resinfor

1 h under gentle agitation. After washing with DMF (6×1 min×2 ml) theFmoc group was removed with 20% piperidine in DMF (1×2 min×2 ml and 2×10min×2 ml) and the resin washed with DMF (6×1 min×2 ml) before thepeptide was extended 2× with N-α-Fmoc-L-aspartic acid G-tert-butyl ester(148 mg, 0.36 mmol) and 4,4-bis(4-hydroxyphenyl)valeric acid (103 mg,0.36 mmol) in the indicated order using the same coupling(HBTU/HOBt-H₂O/DIPEA) and Fmoc-deprotection (20% piperidine in DMF)conditions mentioned before. After the last peptide coupling step, asolution of CuI (2.3 mg, 0.01 mmol), TBTA (6.4 mg, 0.01 mmol) and alkyne10 (99 mg, 0.36 mmol) in a mixture of DMF (1 ml) and THF (1 ml) wasprepared and reacted with the resin at 22° C. for 2 h. After washingwith DMF (6×1 mmx 2 ml), the compound was cleaved by agitating the resinwith a mixture of TFA (4.5 ml), TIPS (250 μl) and H₂O (250 μl) at 22° C.for 2 h. The resin was washed with TFA (1×5 min×2 ml) and the combinedcleavage and washing solutions added drop-wise to ice cold diethyl ether(50 ml). The precipitate was collected by centrifugation and the productpurified by reversed-phase HPLC (95% A/5% B to 20% A/80% B over 30 min).After lyophilization the title compound was collected as a white powder(49 mg, 46 μmol, 38% yield).

1H-NMR (500 MHz, DMSO-d₆): δ 13.01 (s, 1H), 9.19 (br. s, 2H), 8.33 (s,2H), 8.19 (d, J=8.0, 1H), 8.09 (d, J=7.9, 1H) 7.91 (d, J=8.1, 1H), 7.88(t, J=6.0, 1H), 7.84 (s, 1H), 7.79 (br. s, 3H), 6.92 (d, J=8.4, 4H),6.64 (d, J=8.4, 4H), 4.54-4.44 (m, 2H), 4.24 (t, J=7.2, 2H), 4.17 (1d,J=8.3, 5.5, 1H), 3.58 (t, J=5.3, 2H), 3.56-3.50 (m, 4H), 3.38 (t, J=6.1,2H), 3.24-3.15 (m, 2H), 2.97 (sext, J=5.6, 2H), 2.65 (t, J=7.5, 2H),2.64-2.55 (m, 4H), 2.51-2.41 (m, 2H), 2.17 (t, J=8.2, 2H) 1.94 (quin,J=7.5, 2H), 1.88-1.82 (m, 2H), 1.75 (quin, J=7.5, 2H), 1.66-1.60 (m,1H), 1.53-1.46 (m, 1H), 1.45 (s, 3H), 1.28-1.17 (m, 2H). 13C-NMR (125MHz, DMSO-d₆): δ 172.84, 172.79, 172.30, 172.04, 171.62, 169.19, 169.09,164.33, 161.09, 154.96, 146.06, 139.65, 139.58, 127.81, 121.84, 114.68,69.68, 69.46, 68.85, 66.70, 52.34, 49.08, 48.70, 43.86, 38.70, 38.53,37.14, 36.95, 36.75, 34.27, 31.36, 31.27, 29.44, 27.39, 24.42, 24.23,22.28. HRMS (ESI): m/z calcd. for C45H63N12O15S2 [M+H]⁺: 1075.3972;found: 1075.3966.

N-[4,4-bis(4-hydroxyphenyl)pentanoyl]-β-aspartyl-β-aspartyl-N-[2-(2-{2-[(5-{4-[(6E)-6-{(2E)-2-[3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene]ethylidene}-2-{(E)-2-[3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium-2-yl]ethenyl}cyclohex-1-en-1-yl]phenyl}pentanoyl)amino]ethoxy}ethoxy)ethyl]-6-(4-{4-oxo-4-[(5-sulfamoyl-1,3,4-thiadiazol-2-yl)amino]butyl}-1H-1,2,3-triazol-1-yl)-L-norleucinamide(C5c)

To C5a (161 μg, 150 nmol) in DMSO (16.1 μl) was added IRDye

750 NHS ester (99 μg, 83 nmol) in DMSO (10 μl) followed by DMF (100 μl)and DIPEA (2 μl, 12 μmol). The solution was stirred at 22° C. for 2 hand then quenched with sodium hydrogen carbonate (100 μl, 100 mM, pH8.0) before purifying over reversed-phase HPLC (95% A/5% B to 40% A/60%B over 30 min). Fractions containing dye conjugate were identifiedthrough their characteristic UV/VIS spectrum (λ_(max)=756 nm), pooled,lyophilized and dissolved in DMSO (50 d) to give a dark green stocksolution. Its concentration and the reaction yield were determined bymeasuring the absorbance at 756 nm (0756=260′000 M-1 cm-1) of stocksamples diluted 1:200 into PBS (pH 7.4): 1.00 mM, 50 nmol, 60% yield.HRMS (Dual MALDI/ESI): m/z calcd. for C94H121N14028S6 [M+]: 2085.6793;found: 2085.6793.

The binding affinities of the synthetic compounds of FIG. 16 were thencharacterized by fluorescence polarization and by surface plasmonresonance, as follows.

Affinity Determination of CAIX Ligands by Fluorescence Polarization (FP)Measurements.

Fluorescein labelled ligands (5 nM diluted with PBS from DMSO stocks,final DMSO content adjusted to 0.001%) were incubated at 22° C. for 1 hin a black 384-well plate (Greiner, non-binding) in PBS (pH 7.4) withincreasing concentrations of CAIX to a final volume of 60 μl. Thefluorescence anisotropy was measured on a Spectra Max Paradigm multimodeplate reader (Molecular Devices). Experiments were performed intriplicate and the mean anisotropy values fitted to the followingequation using KaleidaGraph 4.1.3 (Synergy Software),

$A = {\frac{1}{2}\left\{ {\left( {\lbrack P\rbrack_{0} + \lbrack L\rbrack_{0} + K_{D}} \right) - \sqrt{\left( {\lbrack P\rbrack_{0} + \lbrack L\rbrack_{0} + K_{D}} \right)^{2} - {{4\lbrack P\rbrack}_{0}\lbrack L\rbrack}_{0}}} \right\}}$

where A is the anisotropy. [P]0 the total protein concentration, [L]0the total concentration of the fluorescently labelled ligand and AD thedissociation constant.

Affinity Determination of CAIX Ligands by Surface Plasmon Resonance(SPR) Measurements.

Surface plasmon resonance experiments were carried out at roomtemperature (25° C.) using a Biacore™ T200 instrument and CM5 chips (GEHealthcare). For all measurements, a PBS buffer (pH 7.4) containing DMSO(5% v/v) and P20 surfactant (0.05% v/v, GE Healthcare) was used. CAIXprotein was immobilized on the chip at about 3,000 response units usingEDC·HCl and NHS as described by the instrument manufacturer. Serialdilutions of unlabelled compounds (0.08 nM to 620 nM in steps of 1/2)were used as analytes. After each cycle, the sensor surface wasregenerated by a short treatment with DMSO (50% v/v) in H₂O. Sensorgramswere solvent corrected and the binding kinetics were analysed with theBiacore™ T200 evaluation software (version 2.0) using a 1:1 Langmuirbinding model.

The best binders featured an Asp-Asp moiety in the linker (C5a and C5b)and a K_(d) value of 0.2±0 1 nM by fluorescence polarization insolution. SPR measurements gave slightly higher dissociation constants.The best binder appeared to be the one with the longest linker.

The binding properties of the best A-493/B-202 conjugate were furtherstudied on SK-RC-52 human kidney cancer cells by fluorescence-activatedcell sorting. For this purposes the fluorescein moiety was replaced witha fluorescent near-infrared dye (IRDye

750). Compounds lacking the B moiety or both A/B moieties were used ascontrols in the experiment.

Cell Culture.

SK-RC-52 and HEK cells were maintained in RPMI medium (Invitrogen)supplemented with fetal calf serum (10% v/v, FCS, Life Technologies) andAntibiotic-Antimycotic (AA, Life Technologies) at 37° C. and 5% CO₂. Forpassaging, cells were detached using Trypsin-EDTA 0.05% (LifeTechnologies) when reaching 90% confluence and re-seeded at a dilutionof 1:10.

Ligand Binding Analysis by Flow Cytometry.

Cells were detached from culture plates using EDTA (50 mM) solution inPBS (pH 7.4), counted and suspended to a final concentration of 1.5×10⁶cells ml-1 in a solution of FCS (1% v/v)/PBS (pH 7.4). Aliquots of 3×10⁵cells (200 μl) were spun down and resuspended in solutions of IRDye

750 (Licor) labelled ligands (30 nM) in FCS (1% v/v) in PBS (200 μl, pH7.4) and incubated at 4° C. for 1 h. Cells were washed once with 200 μlFCS (1% v/v)/

PBS (pH 7.4), spun down, resuspended in a solution of propidium iodide(30 μM. Sigma-Aldrich) in FCS (1% v/v)/PBS (300 μl, pH 7.4) and analysedon a FACS Canto flow cytometer (BD Bioscience). FlowJo Version 8.7(Treestar) was used for data analysis and visualization.

These experiments showed that the IRDye 750 labeled compound C5c (FIG.17 ) stained cells more strongly than the corresponding IRDye 750labeled acetazolamide control C1a (FIG. 17 ). These results are shown inFIG. 18 .

In Vivo Binding Studies

For IVIS imaging experiments, mice bearing subcutaneous SK-RC-52 tumorswere injected intravenously with 3 nmol IRDye

750 labelled CAIX ligands C1c, C5c and C6 (FIGS. 15,17 ) dissolved in 5%v/v DMSO in PBS pH 7.4 (150 μL). Mice were anesthetized with isofluraneand fluorescence images acquired on an IVIS Spectrum imaging system(Xenogen, exposure 1s, binning factor 8, excitation at 745 nm, emissionfilter at 800 nm, f number 2, field of view 13.1). Food and water wasgiven ad libitum between measurements. Mice were subsequently sacrificedby cervical dislocation. Heart, lung, kidney, liver, spleen, a sectionof the intestine, skeletal muscle and the tumour were extracted andimaged individually using above parameters.

The untargeted dye C6 did not preferentially localize to the tumor atany time point, in full analogy to conventional chemotherapeutic agents.The acetazolamide derivative C1c exhibited a rapid preferentialaccumulation in the tumor, but gradually dissociated from the neoplasticmass over time. By contrast, the high-affinity bidentate A-493/B-202ligand C5c exhibited a selective and long-lasting tumor targeting. Thetumor targeting efficiency of C5c and C1c (18% and 3.7% injected doseper gram of at 24 h, respectively) favourably compared to thebiodistribution data obtained in the same animal model using twohigh-affinity human monoclonal antibodies in IgG format.

Preparation of a Radiolabelled Ligand Having CAIX Binding Property

An anti-CAIX ligand having the following chemical structure:

was radiolabelled with Technetium 99m as follows. 50 μL ligand (1.2 mM)in degassed PBS pH 7.4 was mixed with 50 μL SnCl₂ (4 mg/mL) freshlyprepared solution in degassed MQ water, 100 μL Na glucoheptonate (200mg/mL) freshly prepared in degassed MQ water, and 600 μL TBS pH 7.4. Thesolution was degassed for at least 5 min by bubbling nitrogen. 200 μL^(99m)Tc generator eluate (ca. 200 MBq) was added to the solution, whichwas then heated to 90° C. for 20 min and left to cool to roomtemperature.

Evaluation of Biodistribution of Radiolabelled Anti-CAIX Ligand

The biodistribution performance of the Technetium 99m labelled ligand inmice was assessed as follows. Balb/c nu/nu mice were injectedsubcutaneously with 10⁷ SKRC52 renal cell carcinoma cells. EstablishedSKRC52 tumors were allowed to grow to an average size of 500 mm³ beforereceiving intravenous injections of the the radiolabelled ligand. Anuntargeted/irrelevant ligand was also radiolabelled with Technetium 99mand used as negative control.

Six hours after injection mice were sacrificed, individual organs wereexcised and analyzed for radiolabelled ligand uptake.

The results expressed as Injected dose per gram of tissue are shown inFIG. 19 It can be seen that the CAIX ligand strongly localized in thetumor as compared to the untargeted ligand.

Synthesis of an Auristatin Drug Conjugated to a Small Molecule CAIXBinding Moiety Having a Peptide Linker that is Cleavable by Cathepsin B

The reaction scheme and the structure of the drug conjugate of thisexample according to the present invention are shown in FIG. 19 .

The peptide AAZ-triazole-AspArgAspCys-COOH (SEQ ID NO: 6) (1) wasprepared as described previously (Krall et. al., Angew. Chem. Int. Ed.2014, 53, 4231). A solution of 1 (4.5 mg, 5 μmol) in degassed PBS pH 7.4(1 mL) was added to commercially available Maleimido Caproyl ValineCitrulline Para-Amino Benzyl carbamate of Mono Methyl Auristatin E(MC-VC-PAB-MMAE (2), 6.5 mg, 4.9 μmol) and allowed to stand at roomtemperature for 5 min. MMAE is the toxic moiety. MC-VC-PAB is thecleavable linker.

The mixture was purified over HPLC (Synergi RP Polar, 5% MeCN in 0.1%aq. TFA to 80% over 20 min) and product containing fractions identifiedby low-resolution mass spectrometry. After lyophilization the productwas collect as a white powder (7.5 mg, 3.4 μmol, 68%). HRMS:(m/z)[M+2H⁺] C₉₈H₁₅₃N₂₅O₂₈S₃, 1112.0234; found 1112.0237.

Evaluation of the Antitumor Activity of the Auristatin Conjugate withCAIX Binding Moiety and Cathepsin B-Cleavable Peptide Linker.

The antitumor activity of the drug conjugate according to the presentinvention as shown in FIG. 19 was evaluated as follows. The results areshown graphically in FIG. 20 .

Balb/c nu/nu mice were injected subcutaneously with 10⁷ SKRC52 renalcell carcinoma cells. Established SKRC52 tumors were allowed to grow toan average size of 700 mm³ before receiving intravenous injections ofthe SMDC at the following doses and schedules: 50 nm on day 1 only; 25nm each on day 1 and day 2; and 10 nm each on days 1, 2, 3, 4 and 5.

Tumor volumes were recorded daily with the aid of a digital caliper Asignificant antitumor activity was observed even at the lowest dose of10 nmoles.

FIG. 20 shows that a strong antitumor activity of the SMDC was observedin the SKRC52 renal cell carcinoma model established in nude mice. Theregression of tumors with the size of 700 mm³ was observed withdifferent doses and treatment regimes.

All patent documents and other references cited herein are expresslyincorporated herein by reference.

The above embodiments of the invention have been described for thepurpose of illustration only. Many other embodiments falling within thescope of the accompanying claims will be apparent to the skilled reader.

REFERENCES

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1-32. (canceled)
 33. A pharmaceutical composition comprising a targetedtherapeutic agent comprising a terminal group of Formula (I):

conjugated to said therapeutic agent in a pharmaceutically acceptablecarrier, diluent or excipient, wherein said therapeutic agent is linkedto Formula I via a linker that undergoes cleavage in vivo and releasessaid therapeutic agent in an active form at a tumor which expressesCAIX.
 34. The targeted therapeutic agent of claim 33, wherein the linkercomprises a peptide that is cleavable by a protease that is present inthe extracellular matrix of a tumor or that is released after tumor celldeath.
 35. The targeted therapeutic agent of claim 34 wherein saidprotease is selected from the group consisting of MMP-1, MMP-2, MMP-3,Cathepsin A, Cathepsin B, and Cathepsin C.
 36. The targeted therapeuticagent of claim 35, wherein the linker comprises valine-citrulline and iscleavable by Cathepsin B.
 37. The targeted therapeutic agent of claim33, wherein the linker further comprises a self-immolating spacer. 38.The targeted therapeutic agent of claim 33, wherein therapeutic agent isa chemotherapeutic agent selected from an auristatin, a DNA minor groovebinding agent, a DNA minor groove alkylating agent, an enediyne, alexitropsin, a duocarmycin, a topoisomerase inhibitor, a taxane, apuromycin, a dolastatin, a maytansinoid and a vinca alkaloid or acombination of two or more thereof.
 39. The targeted therapeutic agentof claim 33, wherein therapeutic agent is a chemotherapeutic agentselected from Erlotinib (TARCEVA®), Bortezomib (VELCADE®), Fulvestrant(FASLODEX®), Sutent (SU11248), Letrozole (FEMARA®), Imatinib mesylate(GLEEVEC®), PTK787/ZK 222584, Oxaliplatin (Eloxatin®), 5-FU(5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®),Lapatinib (GSK572016), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006),and Gefitinib (IRESSA®), AG1478, AG1571 (SU 5271; Sugen) or acombination of two or more thereof.
 40. The targeted therapeutic agentof claim 33, wherein therapeutic agent is a DNA intercalating agentselected from one or more of acridines, actinomycins, anthracyclines,benzothiopyranoindazoles, pixantrone, crisnatol, brostallicin, CI-958,doxorubicin, actinomycin D, daunorubicin (daunomycin), bleomycin,idarubicin, mitoxantrone, cyclophosphamide, melphalan, mitomycin C,bizelesin, etoposide, mitoxantrone, SN-38, carboplatin, cis-platin,actinomycin D, amsacrine, DACA, pyrazoloacridine, irinotecan andtopotecan and pharmaceutically acceptable salts, acids, derivatives orcombinations of two or more of any of the above.
 41. A targeted smalldrug conjugate of Formula II, comprising a terminal group of Formula I,and a linker to a cytotoxic payload, having the structure of Formula II,

in a pharmaceutically acceptable carrier, diluent or excipient.
 42. Thetargeted pharmaceutical composition of claim 41, wherein said cytotoxicpayload remains attached to said binding moiety while in circulation butis released from said SMDC at a tumor site.
 43. The targetedpharmaceutical composition of claim 41 which causes shrinkage of solidtumors expressing CAIX, selected from glioblastoma, head and neckcancer, lung cancer, cervical cancer, colorectal cancer, breast cancer,and renal cell carcinoma.
 44. The targeted pharmaceutical composition ofclaim 41, wherein solid tumor is a renal carcinoma.
 45. The targetedpharmaceutical composition of claim 41, wherein said conjugate issubstantially stable to hydrolysis in phosphate buffered saline (PBS) at37° C.
 46. The targeted pharmaceutical composition of claim 41, whereinthe linker comprises a peptide that is cleavable by a protease that ispresent in the extracellular matrix of a tumor or that is released aftertumor cell death.
 47. The targeted pharmaceutical composition of claim41, which diffuses out of blood vessels in seconds followingadministration.
 48. A method for treating a CAIX expressing tumor in asubject in need thereof, comprising administration of an effectiveamount of the pharmaceutical composition of claim 33, saidadministration causing regression or shrinkage of said tumor.
 49. Amethod for treating a CAIX expressing tumor in a subject in needthereof, comprising administration of an effective amount of thepharmaceutical composition of claim 41 said administration causingregression or shrinkage of said tumor.
 50. The method of claim 49,wherein said CAIX expressing tumor is selected from glioblastoma, headand neck cancer, lung cancer, cervical cancer, colorectal cancer, breastcancer, and renal cell carcinoma.
 51. The method of claim 50, whereinsaid CAIX expressing tumor is a renal carcinoma.